Decreased Hemolysis and Improved Platelet Function in Blood Components Washed With Plasma-Lyte A Compared to 0.9% Sodium Chloride

Decreased Hemolysis and Improved Platelet Function in Blood Components Washed With Plasma-Lyte A... Abstract Objectives Washing cellular blood products is accepted to ameliorate repeated severe allergic reactions but is associated with RBC hemolysis and suboptimal platelet function. We compared in vitro hemolysis and platelet function in blood components after washing with Plasma-Lyte A (PL-A) vs normal saline (NS). Methods RBC (n = 14) were washed/resuspended in NS or PL-A. Free hemoglobin and heme were determined at 0, 24, 48, and 72 hours. Platelet concentrates (PCs; n = 21) were washed with NS or PL-A and resuspended in same washing solution (n = 13) or ABO-identical plasma (n = 8). Platelet aggregation and spreading were evaluated. Results The 24-hour free hemoglobin and heme levels were higher in NS (P < .05). Improved platelet function was observed in PL-A-washed PCs (P < .001). Discussion PL-A showed less RBC hemolysis and better platelet function than NS. Whether such differences would occur in vivo is unknown. Plasma-Lyte A, Normal saline, Platelet function, Blood products, Hemolysis, Transfusion reactions Transfusions are one of the most common procedures performed in hospitals,1 yet the frequent adverse events of transfusion are often not recognized. These adverse events include transfusion-related acute lung injury, transfusion-associated graft-versus-host disease, transfusion-associated circulatory overload, immunomodulation, inflammation, nosocomial infection, and thrombosis.2,3 While some of these adverse effects are quite rare, others are likely more common but difficult to identify. Transfusion of RBCs and/or platelets is associated, in a dose-dependent fashion, with high morbidity and mortality rates.4-8 It is possible that this observation is related to confounding by differential severity of illness of recipients and acute blood loss. However, mounting evidence indicates that there is direct toxicity from these transfusions, stemming from degradation of these products during storage, known as the “storage lesion,”9-11 and the allogeneic nature (immunologic incompatibility) of the cells and plasma. In a systematic literature review, prolonged RBC storage was found to be associated with adverse clinical outcomes, including mortality and organ failure, based on strong laboratory evidence and observational studies.12 It is uncertain whether this association is related to hemolysis or other storage-induced lesions, and it has not been mitigated by transfusion of shorter-storage RBCs in randomized trials. Some of the deleterious effects appear to be mitigated by modifications such as leukoreduction3,13 and/or saline washing14,15 or avoided by use of autologous transfusions16 and restrictive transfusion practices.17,18 Pretransfusion washing of stored RBC and platelet units is an accepted and valuable therapeutic option for patients with repeated and/or severe allergic or febrile transfusion reactions. Recent data from randomized and observational clinical trials demonstrate that pretransfusion washing reduces inflammatory and immunologic complications, including recurrence of acute leukemia,15 elevations of interleukin-6 and C-reactive protein after pediatric cardiac surgery,14 transfusion-related acute lung injury, and transfusion-associated circulatory overload.13 However, washing of RBCs may not be completely benign. Increased hemolysis may occur in vitro during the washing process, and this may be particularly important for RBCs of longer storage duration, which was associated with a higher complication rate in post hoc analysis of a randomized trial.14,19,20 Suboptimal platelet function has also been observed in washed platelets.21 Cholette et al14 demonstrated increased morbidity in children following cardiac surgery with cardiopulmonary bypass who received washed RBCs that were previously stored for more than 28 days compared to less than 14 days. This important finding suggests that the benefit of washed RBC transfusions may vary depending on the storage duration prior to washing. Current washing techniques and dilution of RBCs and platelets for transfusion employ normal saline (NS; 0.9% sodium chloride, NaCl). Data analysis of transfusions at our medical center in 2016 reveal that we transfused about 4,600 platelet doses, of which 27% were NS-washed platelets, and about 15,000 RBC, of which 12% were NS-washed RBCs. Our washing protocol is indicated for patients with documented IgA deficiency, history of severe allergic transfusion reaction, hematologic malignancies, intrauterine transfusions, newborns, neonatal alloimmune thrombocytopenic purpura, priming the extracorporeal membrane oxygenation circuit for infants younger than 1 year of age, neonates with positive circulating maternal isoagglutinin (anti-A/B) testing, patients expressing polyagglutination, liver transplant (two RBC units for initial cooler), pediatric cardiac surgery cases on cardiopulmonary bypass and 5 years old or younger (first RBC unit washed), and for intrauterine and neonatal exchange transfusions. We evaluated whether washing cellular blood components with a more physiologic solution, Plasma-Lyte A (PL-A), might cause less damage to RBCs and better preserve platelet function as compared with NS.22,23 Materials and Methods Washing Solution Compositions Normal Saline NS is an isotonic 0.9% NaCl solution that is composed of 154 mEq/L Na+ and 154 mEq/L Cl– (354.2 mg/dL Na+ and 546.7 mg/dL Cl–) with a neutral pH of 7. However, data showed that the actual pH of NS preparations for infusion is acidic (pH of 5.5-6) at ambient temperature. The acidity of NS is claimed to be due to the plastic pouch container, which is typically composed of polyvinylchloride.24 Plasma-Lyte A PL-A (Baxter Healthcare Corporation, Deerfield, IL) is an isotonic solution containing 140 mEq/L Na+, 5 mEq/L K+, 3 mEq/L Mg+2, 98 mEq/L Cl–, 27 mEq/L sodium acetate trihydrate (C2H3NaO2-3H2O), and 23 mEq/L sodium gluconate (C6H11NaO7). The pH is adjusted with sodium hydroxide to 7.4 (range of 6.5 to 8.0) Table 1.25 Table 1 Characteristics of Normal Saline and Plasma-Lyte A Washing Solutions   Normal Saline  Plasma-Lyte A  Sodium (mEq/L)  154  140  Potassium (mEq/L)  0  5  Calcium (mEq/L)  0  0  Magnesium (mEq/L)  0  3  Chloride (mEq/L)  154  98  Lactate (mEq/L)  0  0  Gluconate (mEq/L)  0  23  Acetate (mEq/L)  0  27  Osmolarity (mOsm/L)  308  294  pH  5.5  7.4    Normal Saline  Plasma-Lyte A  Sodium (mEq/L)  154  140  Potassium (mEq/L)  0  5  Calcium (mEq/L)  0  0  Magnesium (mEq/L)  0  3  Chloride (mEq/L)  154  98  Lactate (mEq/L)  0  0  Gluconate (mEq/L)  0  23  Acetate (mEq/L)  0  27  Osmolarity (mOsm/L)  308  294  pH  5.5  7.4  View Large Table 1 Characteristics of Normal Saline and Plasma-Lyte A Washing Solutions   Normal Saline  Plasma-Lyte A  Sodium (mEq/L)  154  140  Potassium (mEq/L)  0  5  Calcium (mEq/L)  0  0  Magnesium (mEq/L)  0  3  Chloride (mEq/L)  154  98  Lactate (mEq/L)  0  0  Gluconate (mEq/L)  0  23  Acetate (mEq/L)  0  27  Osmolarity (mOsm/L)  308  294  pH  5.5  7.4    Normal Saline  Plasma-Lyte A  Sodium (mEq/L)  154  140  Potassium (mEq/L)  0  5  Calcium (mEq/L)  0  0  Magnesium (mEq/L)  0  3  Chloride (mEq/L)  154  98  Lactate (mEq/L)  0  0  Gluconate (mEq/L)  0  23  Acetate (mEq/L)  0  27  Osmolarity (mOsm/L)  308  294  pH  5.5  7.4  View Large Washed Red Blood Cells RBC units (n = 14) were procured randomly from the blood bank at Strong Memorial Hospital/University of Rochester Medical Center. A prewash sample (12 mL) was collected from each RBC unit as a baseline using a syringe and needle-free sampling site coupler. The prewash hemoglobin and hematocrit were tested using a Sysmex KX-21N cell counter (Sysmex America, Mundelein, IL). Each RBC unit (10 AS-1, 3 AS-3, and 1 CPD-A1) was divided into two equal parts (150 ± 10 mL) using aseptic technique into a 600-mL Blood Transfer Unit Pack with piercing pin (Charter Medical, Winston-Salem, NC). Each half RBC unit was then washed employing a routine clinical protocol on a COBE 2991 Blood Cell Processor (Terumo, Lakewood, CO)26 with 1 L of either NS or PL-A as a wash solution. After the wash solution was added to the RBCs, in an open system method that allows storage for up to 24 hours, a supernatant consisting of the wash solution, plasma proteins, and anticoagulant/preservative was separated from the RBCs by centrifugation. The supernatant was then expressed into a waste bag. The process was repeated for a total of three washing cycles. Once removed from the blood processor, the washed RBCs were resuspended with 20 mL of the wash solution with the syringe and needle-free sampling site coupler. Final volumes and hematocrits for NS and PL-A averaged 87.0 ± 16.4 mL, 61.1%, and 86.7 ± 14.6 mL, 58.8%, respectively. Washed RBCs were maintained at 1°C to 6°C. Samples (12 mL each) were collected from each washed product immediately after washing/resuspending, and at 24, 48, and 72 hours post washing. Cell counts were performed at each time point using a Sysmex KX-21N analyzer. Samples were then immediately centrifuged in a Sorvall RC-3 centrifuge (Beckman Coulter, Carlsbad, CA) for 15 minutes at 4300g. Supernatants were dispensed into labeled cryovials and frozen at –80°C until further testing. Quantification of free heme and free hemoglobin in supernatants was performed using QuantiChrom Assay Kits (Bioassay Systems, Hayward, CA) as previously described.27 Washed Platelets Platelet-rich plasma (PRP) samples were prepared from whole blood and aliquoted (3 mL) from clinical grade platelet concentrates (PC) units (storage period of 5 days) as prepared for transfusion (n = 21). Samples were then washed via an in vitro tube model similar to our standard clinical washing protocol for the COBE 2991.28 Briefly, samples were centrifuged for 10 minutes at 1200g, resuspended in 4.5 mL of either NS or PL-A solution, centrifuged again, and resuspended in 3 mL of the corresponding wash solution (n = 13) or ABO-identical fresh plasma (n = 8). Platelet aggregation studies were performed with light transmission aggregometry on PRP samples (average platelet count 255 ± 27 × 109/L) as per manufacturer’s instructions on a lumiaggregometer (560VS, Chrono-Log Corporation, Havertown, PA) as previously described.29 Adenosine diphosphate (ADP, 10 µmol/L), epinephrine (10 µmol/L), and collagen (2 µg/mL) agonists (Chrono-log Corporation) were added simultaneously to induce platelet aggregation. ADP and epinephrine were added at time 0 followed by collagen at 10 minutes. Platelet aggregations were then quantified over 20 minutes. Washed Platelet Spreading Assay To test the effects of washing and washing fluids on platelet spreading, which is an indication of platelet adherence function, four additional stored PC samples (storage length of 5 days) and four fresh platelet-rich plasma sample controls were washed and spread on fibrinogen-coated coverslips as previously described.29 Briefly, fresh whole blood samples were collected in 3.2% sodium citrate vacutainer tubes from healthy donors who were free of platelet inhibiting medications. Samples were processed immediately and 3 mL of PRP was isolated through centrifugation (10 minutes at 160g). Samples were then washed as described above with either NS or PL-A. Stored PC samples were obtained and washed as described above in both solutions. Platelets were diluted to a final concentration of 33 × 109 platelets/L with respective washing solutions. The platelets were then incubated at 37°C for 45 minutes on glass coverslips that had been coated with 0.25 mg/mL fibrinogen for 2 hours and blocked with 0.5% bovine serum albumin. Unbound platelets were washed away from coverslips with phosphate-buffered saline (PBS) and bound platelets were fixed with 4% paraformaldehyde for 15 minutes and again washed with PBS before being mounted onto glass slides. Platelet spreading was visualized by light microscopy (Zeiss Axiovert 200, Carl Zeiss, Thornwood, NY) and three or four fields were photographed. Platelets were counted and categorized based on their level of spreading by a single technician blinded to the washing solution. Platelets were categorized as (1) not spread if they lacked filopodia, (2) partially spread if they had filopodia but lacked lamellipodia, and (3) fully spread if they demonstrated lamellipodia.29 Statistical Analysis Data are reported as means ± standard error of the mean (SEM) in RBC experiments. Comparisons were performed between RBCs washed with NS vs PL-A using Wilcoxon signed-rank test. Data of the free hemoglobin concentrations of shorter- and longer-storage RBC washed with either NS or PL-A are reported as means ± standard deviation (SD) and the two-tailed Student t tests is used for comparisons. Means ± SD is also used in platelet experiments. Platelet aggregation of platelets washed in NS and PL-A were compared for resuspension with either the washing solution or ABO-identical plasma using two-tailed Student t tests. t tests were also used for platelet spreading experiments where fresh platelet were added to both washing solutions for direct comparisons. A P value of ≤.05 was considered statistically significant. Results Washing With Plasma-Lyte A Leads to Reduced Hemolysis Compared With Normal Saline The median storage age of RBC units was 19 days (range, 10-39 days). The mean ± SEM prewashed hemoglobin and hematocrit were 19.3 ± 0.41 mg/dL and 58.8% ± 1.02%, respectively. Immediately before and after washing, RBC supernatants from both arms had similar mean free hemoglobin and heme Figure 1. Washing reduced both free hemoglobin and free heme by about 37% in NS and 43% in PL-A group, respectively: free hemoglobin (mg/dL) prewash 83.7 ± 15, NS postwash 54 ± 3.5, PL-A postwash 48 ± 3.7 (P = .07); free heme (µmol/L) prewash 46 ± 8.4, NS postwash 29 ± 1.7, PL-A postwash 26 ± 2.2 (P = .28) (Figure 1). Figure 1 View largeDownload slide Free hemoglobin and heme concentrations in washed RBC. Changes in the supernatant concentrations of free hemoglobin (A) and free heme (B) of RBCs washed with 0.9% NaCl (NS) or Plasma-Lyte A (PL-A) over 72 hours. Data are mean ± standard error of the mean. Pre, prewash; Post, postwash. Figure 1 View largeDownload slide Free hemoglobin and heme concentrations in washed RBC. Changes in the supernatant concentrations of free hemoglobin (A) and free heme (B) of RBCs washed with 0.9% NaCl (NS) or Plasma-Lyte A (PL-A) over 72 hours. Data are mean ± standard error of the mean. Pre, prewash; Post, postwash. However, by 24 hours of storage at 1°C to 6°C after washing (the current limit for clinical use in an open system such as the COBE 2991 cell washer), supernatant free hemoglobin and heme levels were significantly increased in RBCs stored in NS as compared to PL-A: free hemoglobin (mg/dL): NS 114 ± 11, PL-A 74 ± 5.7 (109% increase; P < .001); free heme (µmol/L): NS 63 ± 6.1, PL-A 41 ± 3.5 (117% increase; P < .001). These differences between use of NS and PL-A increased over storage time, indicating that prolonged storage after NS washing predisposed to greater hemolysis than PL-A washing (Figure 1). Plasma-Lyte A Mitigates Hemolysis After Washing of Longer Storage Duration RBCs Substantial differences were detected in the free hemoglobin and free heme levels between the shorter-storage RBC units (n = 5; mean storage duration, 14.2 ± 3 days) and longer-storage RBC units (n = 4; mean storage duration, 34 ± 3.8 days, P < .001). Although not statistically significant, the average baseline (before washing) levels of free hemoglobin and free heme were 86% and 99% higher in the longer-storage RBC units than the shorter-storage RBC (free hemoglobin 132 mg/dL vs 71 mg/dL; free heme 75 μmol/L vs 38 μmol/L, respectively) Table 2 and Table 3. No significant changes in the free heme were observed between the shorter-storage and longer-storage RBC units immediately after washing with either NS or PL-A (Table 3). However, a significant increase (P = .025) in the free hemoglobin was observed in the NS-washed longer-storage RBC (Table 2). Substantial increases were seen in the 24-hour free hemoglobin and free heme in the NS-washed longer-storage RBC (43% and 58%, respectively). In the PL-A-washed RBC, in contrast, the 24-hour free hemoglobin and free heme levels were only slightly higher than baseline in the longer-storage RBC (11% and 15%, respectively). These increases in hemolysis were significantly smaller than NS-washed longer-storage RBC units (P = .024 and .012, respectively). Similar patterns were seen in the 48- and 72-hour RBC (Tables 2 and 3). Table 2 Free Hemoglobin Concentrations of Shorter- and Longer-Storage RBC Washed With Normal Saline (NS) or Plasma-Lyte A (PL-A)a Washing Solution  Free Hemoglobin  Shorter Storage RBC  Longer Storage RBC  NS  PL-A  P Valueb  NS  PL-A  P Valueb  Baseline  71 ± 56  —  132 ± 71  —  Immediately after washing  59 ± 19  53 ± 19  .608  55 ± 1.7  40 ± 8  .025  24 hours after washing  85 ± 47  65 ± 18  .418  121 ± 26  72 ± 14  .024  48 hours after washing  101 ± 49  65 ± 21  .186  121 ± 34  94 ± 28  .342  72 hours after washing  137 ± 40  90 ± 18  .055  220 ± 80  142 ± 39  .148  Washing Solution  Free Hemoglobin  Shorter Storage RBC  Longer Storage RBC  NS  PL-A  P Valueb  NS  PL-A  P Valueb  Baseline  71 ± 56  —  132 ± 71  —  Immediately after washing  59 ± 19  53 ± 19  .608  55 ± 1.7  40 ± 8  .025  24 hours after washing  85 ± 47  65 ± 18  .418  121 ± 26  72 ± 14  .024  48 hours after washing  101 ± 49  65 ± 21  .186  121 ± 34  94 ± 28  .342  72 hours after washing  137 ± 40  90 ± 18  .055  220 ± 80  142 ± 39  .148  aData are mean ± standard deviation. bBy the Student t test. View Large Table 2 Free Hemoglobin Concentrations of Shorter- and Longer-Storage RBC Washed With Normal Saline (NS) or Plasma-Lyte A (PL-A)a Washing Solution  Free Hemoglobin  Shorter Storage RBC  Longer Storage RBC  NS  PL-A  P Valueb  NS  PL-A  P Valueb  Baseline  71 ± 56  —  132 ± 71  —  Immediately after washing  59 ± 19  53 ± 19  .608  55 ± 1.7  40 ± 8  .025  24 hours after washing  85 ± 47  65 ± 18  .418  121 ± 26  72 ± 14  .024  48 hours after washing  101 ± 49  65 ± 21  .186  121 ± 34  94 ± 28  .342  72 hours after washing  137 ± 40  90 ± 18  .055  220 ± 80  142 ± 39  .148  Washing Solution  Free Hemoglobin  Shorter Storage RBC  Longer Storage RBC  NS  PL-A  P Valueb  NS  PL-A  P Valueb  Baseline  71 ± 56  —  132 ± 71  —  Immediately after washing  59 ± 19  53 ± 19  .608  55 ± 1.7  40 ± 8  .025  24 hours after washing  85 ± 47  65 ± 18  .418  121 ± 26  72 ± 14  .024  48 hours after washing  101 ± 49  65 ± 21  .186  121 ± 34  94 ± 28  .342  72 hours after washing  137 ± 40  90 ± 18  .055  220 ± 80  142 ± 39  .148  aData are mean ± standard deviation. bBy the Student t test. View Large Table 3 Free Heme Concentrations of Shorter- and Longer-Storage RBC Washed With Either Normal Saline (NS) or Plasma-Lyte A (PL-A)a Washing Solution  Free Heme  Shorter Storage RBC  Longer Storage RBC  NS  PL-A  P Valueb  NS  PL-A  P Valueb  Baseline  38 ± 30  —  75 ± 39  —  Immediately after washing  29 ± 7  28 ± 12  .852  30 ± 6  22 ± 5  .087  24 hours after washing  44 ± 25  35 ± 12  .478  70 ± 13  40 ± 6  .012  48 hours after washing  54 ± 21  37 ± 12  .182  64 ± 9  51 ± 9  .147  72 hours after washing  81 ± 19  48 ± 11  .015  157 ± 40  80 ± 10  .026  Washing Solution  Free Heme  Shorter Storage RBC  Longer Storage RBC  NS  PL-A  P Valueb  NS  PL-A  P Valueb  Baseline  38 ± 30  —  75 ± 39  —  Immediately after washing  29 ± 7  28 ± 12  .852  30 ± 6  22 ± 5  .087  24 hours after washing  44 ± 25  35 ± 12  .478  70 ± 13  40 ± 6  .012  48 hours after washing  54 ± 21  37 ± 12  .182  64 ± 9  51 ± 9  .147  72 hours after washing  81 ± 19  48 ± 11  .015  157 ± 40  80 ± 10  .026  aData are mean ± standard deviation. bBy the Student t test. View Large Table 3 Free Heme Concentrations of Shorter- and Longer-Storage RBC Washed With Either Normal Saline (NS) or Plasma-Lyte A (PL-A)a Washing Solution  Free Heme  Shorter Storage RBC  Longer Storage RBC  NS  PL-A  P Valueb  NS  PL-A  P Valueb  Baseline  38 ± 30  —  75 ± 39  —  Immediately after washing  29 ± 7  28 ± 12  .852  30 ± 6  22 ± 5  .087  24 hours after washing  44 ± 25  35 ± 12  .478  70 ± 13  40 ± 6  .012  48 hours after washing  54 ± 21  37 ± 12  .182  64 ± 9  51 ± 9  .147  72 hours after washing  81 ± 19  48 ± 11  .015  157 ± 40  80 ± 10  .026  Washing Solution  Free Heme  Shorter Storage RBC  Longer Storage RBC  NS  PL-A  P Valueb  NS  PL-A  P Valueb  Baseline  38 ± 30  —  75 ± 39  —  Immediately after washing  29 ± 7  28 ± 12  .852  30 ± 6  22 ± 5  .087  24 hours after washing  44 ± 25  35 ± 12  .478  70 ± 13  40 ± 6  .012  48 hours after washing  54 ± 21  37 ± 12  .182  64 ± 9  51 ± 9  .147  72 hours after washing  81 ± 19  48 ± 11  .015  157 ± 40  80 ± 10  .026  aData are mean ± standard deviation. bBy the Student t test. View Large Plasma-Lyte A Preserves Platelet Function as Compared With Saline The baseline platelet count was 1,478 ± 257 × 109/L (mean ± SD). Following washing, the platelet count dropped by about 5% in both groups (1,413 ± 185 × 109/L; P > .05), demonstrating that the type of washing solution had no influence on platelet recovery. As stored platelets exhibit minimal to no platelet aggregation with single agonists,30,31 baseline platelet aggregation of nonwashed stored PCs was performed with a combination of three agonists (ADP, epinephrine, and collagen) and averaged 40 ± 19%. No aggregation was detected in PCs washed and resuspended in NS. In contrast, aggregation of PCs washed and resuspended in PL-A averaged 29 ± 13% (P < .001 in comparison to NS). Resuspension in ABO-identical plasma improved platelet aggregation significantly for PCs washed in NS or PL-A; however, the PL-A arm still showed superior results in comparison to NS washing (P < .05) Table 4. A representative example of PRP platelet aggregation in response to ADP (10 µmol/L) and epinephrine (10 µmol/L) at time 0 and collagen (2 µg/mL) at 10 minutes is shown in Figure 2. While platelet aggregation of two washed PCs with PL-A (traces 3 and 4) were above 50% for both units, washing the same PC units with NS (traces 1 and 2) showed no measurable platelet aggregation (0%) for both units. Table 4 Light Transmission Platelet Aggregation (%) of Platelet Concentrates (PCs) Washed With Normal Saline (NS) or Plasma-Lyte A (PL-A)a   PCs  NS-Washed PCs  PL-A-Washed PCs  P Value  Baseline (n = 21)  40 ± 19  —  —    Resuspension in same wash solution (n = 13)  —  0  29 ± 13  <.001  Resuspension in ABO-identical plasma (n = 8)  —  41 ± 10  52 ± 5  <.05  P value    <.001  <.05      PCs  NS-Washed PCs  PL-A-Washed PCs  P Value  Baseline (n = 21)  40 ± 19  —  —    Resuspension in same wash solution (n = 13)  —  0  29 ± 13  <.001  Resuspension in ABO-identical plasma (n = 8)  —  41 ± 10  52 ± 5  <.05  P value    <.001  <.05    aData are mean ± standard deviation. View Large Table 4 Light Transmission Platelet Aggregation (%) of Platelet Concentrates (PCs) Washed With Normal Saline (NS) or Plasma-Lyte A (PL-A)a   PCs  NS-Washed PCs  PL-A-Washed PCs  P Value  Baseline (n = 21)  40 ± 19  —  —    Resuspension in same wash solution (n = 13)  —  0  29 ± 13  <.001  Resuspension in ABO-identical plasma (n = 8)  —  41 ± 10  52 ± 5  <.05  P value    <.001  <.05      PCs  NS-Washed PCs  PL-A-Washed PCs  P Value  Baseline (n = 21)  40 ± 19  —  —    Resuspension in same wash solution (n = 13)  —  0  29 ± 13  <.001  Resuspension in ABO-identical plasma (n = 8)  —  41 ± 10  52 ± 5  <.05  P value    <.001  <.05    aData are mean ± standard deviation. View Large Figure 2 View largeDownload slide Light transmission platelet aggregation. The percentage of light transmission (0% set at time 0) is shown for two washed platelet concentrates with NS (traces 1 and 2; blue and black, respectively) and PL-A (traces 3 and 4; red and green, respectively) in response to adenosine diphosphate (10 µmol/L) and epinephrine (10 µmol/L) at time 0 (ADP and Epi), and collagen (2 µg/mL) at 10 minutes simultaneously. Arrows show time of applying each agonist. Figure 2 View largeDownload slide Light transmission platelet aggregation. The percentage of light transmission (0% set at time 0) is shown for two washed platelet concentrates with NS (traces 1 and 2; blue and black, respectively) and PL-A (traces 3 and 4; red and green, respectively) in response to adenosine diphosphate (10 µmol/L) and epinephrine (10 µmol/L) at time 0 (ADP and Epi), and collagen (2 µg/mL) at 10 minutes simultaneously. Arrows show time of applying each agonist. Plasma-Lyte A Improves Platelet Spreading as Compared With Normal Saline As shown in Figure 3C, higher percentages of partially and fully spread platelets were seen when PCs were washed with PL-A as compared with NS (P < .001). The highest percentage of platelets adhering to the fibrinogen coverslip but failing to spread were seen in both stored and fresh platelet samples after washing with NS. The highest percentages of fully spread platelets were observed in fresh PRP samples washed with PL-A (P < .001; Figure 3C). Figure 3 View largeDownload slide Platelet spreading assays. A, Differential interference contrast micrographs demonstrate different stages of platelet spreading that were used as criteria for counting and quantification of platelet spreading. Based on the level of spreading, fully spread platelets were identified by the presence of lamellipodia (arrow) (left); partially spread platelets are indicated by the presence of filopodia (arrowhead) but not lamellipodia (center); and platelets that were round and lacked filopodia were considered not spread (right). B, Examples of platelet spreading of fresh platelets washed with 0.9% NaCl (NS) or Plasma-Lyte A (PL-A) and platelet concentrates washed with NS or PL-A. C, Quantification of whole images derived from experimental samples using images from A as a gauge. Data below the bar chart are percentage of total count (mean ± standard deviation). P < .001 in comparison to NS-washed platelet concentrates (*) and NS-washed fresh platelets (**). Figure 3 View largeDownload slide Platelet spreading assays. A, Differential interference contrast micrographs demonstrate different stages of platelet spreading that were used as criteria for counting and quantification of platelet spreading. Based on the level of spreading, fully spread platelets were identified by the presence of lamellipodia (arrow) (left); partially spread platelets are indicated by the presence of filopodia (arrowhead) but not lamellipodia (center); and platelets that were round and lacked filopodia were considered not spread (right). B, Examples of platelet spreading of fresh platelets washed with 0.9% NaCl (NS) or Plasma-Lyte A (PL-A) and platelet concentrates washed with NS or PL-A. C, Quantification of whole images derived from experimental samples using images from A as a gauge. Data below the bar chart are percentage of total count (mean ± standard deviation). P < .001 in comparison to NS-washed platelet concentrates (*) and NS-washed fresh platelets (**). Discussion RBCs stored for 10 to 39 days and then washed with PL-A experience strikingly less hemolysis during short-term postwashing storage compared with those washed and stored in NS. Whether or not similar degrees of hemolysis would occur in vivo after transfusion is not known. Less hemolysis was observed in the PL-A-washed RBCs stored for more than 4 weeks. We speculate this is due to the improved membrane stability of the longer-stored RBCs washed with PL-A. Although 48 to 72-hour storage of washed RBCs is not clinically employed at present in our center, we evaluated these two time points to measure differences between washing solutions at longer storage intervals. Hemolysis after NS as compared with PL-A washing became progressively worse as postwash storage continued. PL-A washing led to improved postwash platelet aggregation and spreading as compared to NS. Whether such differences would occur in vivo is not known, and the clinical implications are yet to be determined. Reducing free hemoglobin and heme may be important in improving outcomes in transfused patients. Circulating free hemoglobin and heme levels have been associated with thrombosis in animal models,32 transfused patients, and patients with sickle cell disease.33,34 NS, as opposed to PL-A, has been linked to increased renal injury and mortality in animal models and patients.35,36 More physiological levels of potassium and sodium were also reported in RBC washed with Plasma-Lyte as compared to NS washed RBC using the same washing device.37 We speculate that hemolysis may be a contributory cause. Our results indicate that washing RBC with PL-A is a superior approach for maintaining RBC integrity and thus potentially safer than NS. These findings support further investigation of whether NS is truly a suitable diluent for RBC transfusion. PL-A washing might also facilitate lengthening the permissible storage period of washed RBCs. In addition to removing most of the metabolic products and mediators that accumulate during storage, the results of our study confirm that washing blood products with PL-A better maintained RBC structural integrity and preserved platelet function as compared to the commonly used washing solution, NS. The remarkable differences in free heme and hemoglobin concentrations detected in the shorter versus longer stored RBCs washed with NS versus PL-A are additional evidence of the protective effect of PL-A on RBC integrity. PL-A also has potential to partially reverse storage-induced platelet dysfunction. This effect was specific to PL-A in both platelet aggregation and platelet spreading assays, because it was not seen when stored platelets were treated with NS (Figure 2 and Figure 3C). Even when washed platelets were resuspended in ABO-identical plasma, platelet function tests were significantly better in the PL-A-washed platelet arm. Of note, PL-A is almost twice as expensive as NS; the additional cost per liter is approximately $1.00. There are, to our knowledge, no other studies examining the effects of saline vs balanced salt solution washing on RBC lysis and platelet function. Our findings are thus novel. These in vitro studies are at present of unknown clinical relevance. Nonetheless, a growing body of data suggest that free hemoglobin, heme, and nontransferrin-bound iron can mediate morbidity and mortality in animal models.38,39 Our new data support the suggestion that further studies are warranted to determine whether PL-A (or similar balanced crystalloid solutions) has clinical outcome benefits compared to NS when used for resuscitation, coadministration with RBCs and platelets, apheresis, and cell washing in patients requiring crystalloid infusion or transfusion. Acknowledgments We thank Ann Casey from Dr Phipps’ laboratory, blood bank/transfusion service staff and supervisors, and hemostasis and thrombosis laboratory staff for their assistance with these studies. References 1. Healthcare Cost and Utilization Project (HCUP). Facts and Figures: Statistics on Hospital-Based Care in the United States, 2009 . Rockville, MD: Agency for Healthcare Research and Quality; 2011. 2. Popovsky MA, Moore SB. Mechanism of transfusion-related acute lung injury. Blood . 1991; 77: 2299. Google Scholar PubMed  3. Tartter PI, Mohandas K, Azar P, et al.   Randomized trial comparing packed red cell blood transfusion with and without leukocyte depletion for gastrointestinal surgery. Am J Surg . 1998; 176: 462- 466. Google Scholar CrossRef Search ADS PubMed  4. Koch CG, Li L, Sessler DI, et al.   Duration of red-cell storage and complications after cardiac surgery. N Engl J Med . 2008; 358: 1229- 1239. Google Scholar CrossRef Search ADS PubMed  5. Aubron C, Syres G, Nichol A, et al.   A pilot feasibility trial of allocation of freshest available red blood cells versus standard care in critically ill patients. Transfusion . 2012; 52: 1196- 1202. Google Scholar CrossRef Search ADS PubMed  6. Wang D, Sun J, Solomon SB, et al.   Transfusion of older stored blood and risk of death: a meta-analysis. Transfusion . 2012; 52: 1184- 1195. Google Scholar CrossRef Search ADS PubMed  7. Lelubre C, Vincent JL. Relationship between red cell storage duration and outcomes in adults receiving red cell transfusions: a systematic review. Crit Care . 2013; 17: R66. Google Scholar CrossRef Search ADS PubMed  8. Solomon SB, Wang D, Sun J, et al.   Mortality increases after massive exchange transfusion with older stored blood in canines with experimental pneumonia. Blood . 2013; 121: 1663- 1672. Google Scholar CrossRef Search ADS PubMed  9. Flegel WA, Natanson C, Klein HG. Does prolonged storage of red blood cells cause harm? Br J Haematol . 2014; 165: 3- 16. Google Scholar CrossRef Search ADS PubMed  10. Kim-Shapiro DB, Lee J, Gladwin MT. Storage lesion: role of red blood cell breakdown. Transfusion . 2011; 51: 844- 851. Google Scholar CrossRef Search ADS PubMed  11. van de Watering L. Red cell storage and prognosis. Vox Sang . 2011; 100: 36- 45. Google Scholar CrossRef Search ADS PubMed  12. Tinmouth A, Fergusson D, Yee IC, et al.  ; ABLE Investigators; Canadian Critical Care Trials Group. Clinical consequences of red cell storage in the critically ill. Transfusion . 2006; 46: 2014- 2027. Google Scholar CrossRef Search ADS PubMed  13. Blumberg N, Heal JM, Gettings KF, et al.   An association between decreased cardiopulmonary complications (transfusion-related acute lung injury and transfusion-associated circulatory overload) and implementation of universal leukoreduction of blood transfusions. Transfusion . 2010; 50: 2738- 2744. Google Scholar CrossRef Search ADS PubMed  14. Cholette JM, Henrichs KF, Alfieris GM, et al.   Washing red blood cells and platelets transfused in cardiac surgery reduces postoperative inflammation and number of transfusions: results of a prospective, randomized, controlled clinical trial. Pediatr Crit Care Med . 2012; 13: 290- 299. Google Scholar CrossRef Search ADS PubMed  15. Blumberg N, Heal JM, Rowe JM. A randomized trial of washed red blood cell and platelet transfusions in adult acute leukemia [ISRCTN76536440]. BMC Blood Disord . 2004; 4: 6. Google Scholar PubMed  16. Zhou J. A review of the application of autologous blood transfusion. Braz J Med Biol Res . 2016; 49: e5493. Google Scholar CrossRef Search ADS PubMed  17. Prescott LS, Taylor JS, Lopez-Olivo MA, et al.   How low should we go: a systematic review and meta-analysis of the impact of restrictive red blood cell transfusion strategies in oncology. Cancer Treat Rev . 2016; 46: 1- 8. Google Scholar CrossRef Search ADS PubMed  18. Seitz KP, Sevransky JE, Martin GS, et al.   Evaluation of RBC transfusion practice in adult ICUs and the effect of restrictive transfusion protocols on routine care. Crit Care Med . 2017; 45: 271- 281. Google Scholar CrossRef Search ADS PubMed  19. Harm SK, Raval JS, Cramer J, et al.   Haemolysis and sublethal injury of RBCs after routine blood bank manipulations. Transfus Med . 2012; 22: 181- 185. Google Scholar CrossRef Search ADS PubMed  20. Masalunga C, Cruz M, Porter B, et al.   Increased hemolysis from saline pre-washing RBCs or centrifugal pumps in neonatal ECMO. J Perinatol . 2007; 27: 380- 384. Google Scholar CrossRef Search ADS PubMed  21. Schoenfeld H, Muhm M, Doepfmer U, et al.   Platelet activity in washed platelet concentrates. Anesth Analg . 2004; 99: 17- 20. Google Scholar CrossRef Search ADS PubMed  22. Verma B, Luethi N, Cioccari L, et al.   A multicentre randomised controlled pilot study of fluid resuscitation with saline or Plasma-Lyte 148 in critically ill patients. Crit Care Resusc . 2016; 18: 205- 212. Google Scholar PubMed  23. Allen CH, Goldman RD, Bhatt S, et al.   A randomized trial of Plasma-Lyte A and 0.9% sodium chloride in acute pediatric gastroenteritis. BMC Pediatr . 2016; 16: 117. Google Scholar CrossRef Search ADS PubMed  24. Story DA, Thistlethwaite P, Bellomo R. The effect of PVC packaging on the acidity of 0.9% saline. Anaesth Intensive Care . 2000; 28: 287- 292. Google Scholar PubMed  25. Noritomi DT, Pereira AJ, Bugano DD, et al.   Impact of Plasma-Lyte pH 7.4 on acid-base status and hemodynamics in a model of controlled hemorrhagic shock. Clinics (Sao Paulo) . 2011; 66: 1969- 1974. Google Scholar CrossRef Search ADS PubMed  26. Smith T, Riley W, Fitzgerald D. In vitro comparison of two different methods of cell washing. Perfusion . 2013; 28: 34- 37. Google Scholar CrossRef Search ADS PubMed  27. Spinelli SL, Lannan KL, Casey AE, et al.   Isoprostane and isofuran lipid mediators accumulate in stored red blood cells and influence platelet function in vitro. Transfusion . 2014; 54: 1569- 1579. Google Scholar CrossRef Search ADS PubMed  28. Kalmin ND, Brown DJ. Platelet washing with a blood cell processor. Transfusion . 1982; 22: 125- 127. Google Scholar CrossRef Search ADS PubMed  29. Refaai MA, Carter J, Henrichs KF, et al.   Alterations of platelet function and clot formation kinetics after in vitro exposure to anti-A and -B. Transfusion . 2013; 53: 382- 393. Google Scholar CrossRef Search ADS PubMed  30. AuBuchon JP, Herschel L, Roger J, et al.   Preliminary validation of a new standard of efficacy for stored platelets. Transfusion . 2004; 44: 36- 41. Google Scholar CrossRef Search ADS PubMed  31. Murphy S. Utility of in vitro tests in predicting the in vivo viability of stored PLTs. Transfusion . 2004; 44: 618- 619; author reply 619. Google Scholar CrossRef Search ADS PubMed  32. Schaer DJ, Buehler PW, Alayash AI, et al.   Hemolysis and free hemoglobin revisited: exploring hemoglobin and hemin scavengers as a novel class of therapeutic proteins. Blood . 2013; 121: 1276- 1284. Google Scholar CrossRef Search ADS PubMed  33. Ballas SK. Sickle cell anemia with few painful crises is characterized by decreased red cell deformability and increased number of dense cells. Am J Hematol . 1991; 36: 122- 130. Google Scholar CrossRef Search ADS PubMed  34. Ballas SK, Marcolina MJ. Hyperhemolysis during the evolution of uncomplicated acute painful episodes in patients with sickle cell anemia. Transfusion . 2006; 46: 105- 110. Google Scholar CrossRef Search ADS PubMed  35. Yunos NM, Bellomo R, Story D, et al.   Bench-to-bedside review: chloride in critical illness. Crit Care . 2010; 14: 226. Google Scholar CrossRef Search ADS PubMed  36. Chowdhury AH, Cox EF, Francis ST, et al.   A randomized, controlled, double-blind crossover study on the effects of 2-L infusions of 0.9% saline and Plasma-Lyte® 148 on renal blood flow velocity and renal cortical tissue perfusion in healthy volunteers. Ann Surg . 2012; 256: 18- 24. Google Scholar CrossRef Search ADS PubMed  37. Sasaki J, Tirotta C, Lim H, et al.   Comparison of stored red blood cell washing techniques for priming extracorporeal circuits. Perfusion . 2018; 33: 130- 135. Google Scholar CrossRef Search ADS PubMed  38. Stapley R, Rodriguez C, Oh JY, et al.   Red blood cell washing, nitrite therapy, and antiheme therapies prevent stored red blood cell toxicity after trauma-hemorrhage. Free Radic Biol Med . 2015; 85: 207- 218. Google Scholar CrossRef Search ADS PubMed  39. Larsen R, Gozzelino R, Jeney V, et al.   A central role for free heme in the pathogenesis of severe sepsis. Sci Transl Med . 2010; 2: 51ra71. Google Scholar CrossRef Search ADS PubMed  © American Society for Clinical Pathology, 2018. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png American Journal of Clinical Pathology Oxford University Press

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Abstract

Abstract Objectives Washing cellular blood products is accepted to ameliorate repeated severe allergic reactions but is associated with RBC hemolysis and suboptimal platelet function. We compared in vitro hemolysis and platelet function in blood components after washing with Plasma-Lyte A (PL-A) vs normal saline (NS). Methods RBC (n = 14) were washed/resuspended in NS or PL-A. Free hemoglobin and heme were determined at 0, 24, 48, and 72 hours. Platelet concentrates (PCs; n = 21) were washed with NS or PL-A and resuspended in same washing solution (n = 13) or ABO-identical plasma (n = 8). Platelet aggregation and spreading were evaluated. Results The 24-hour free hemoglobin and heme levels were higher in NS (P < .05). Improved platelet function was observed in PL-A-washed PCs (P < .001). Discussion PL-A showed less RBC hemolysis and better platelet function than NS. Whether such differences would occur in vivo is unknown. Plasma-Lyte A, Normal saline, Platelet function, Blood products, Hemolysis, Transfusion reactions Transfusions are one of the most common procedures performed in hospitals,1 yet the frequent adverse events of transfusion are often not recognized. These adverse events include transfusion-related acute lung injury, transfusion-associated graft-versus-host disease, transfusion-associated circulatory overload, immunomodulation, inflammation, nosocomial infection, and thrombosis.2,3 While some of these adverse effects are quite rare, others are likely more common but difficult to identify. Transfusion of RBCs and/or platelets is associated, in a dose-dependent fashion, with high morbidity and mortality rates.4-8 It is possible that this observation is related to confounding by differential severity of illness of recipients and acute blood loss. However, mounting evidence indicates that there is direct toxicity from these transfusions, stemming from degradation of these products during storage, known as the “storage lesion,”9-11 and the allogeneic nature (immunologic incompatibility) of the cells and plasma. In a systematic literature review, prolonged RBC storage was found to be associated with adverse clinical outcomes, including mortality and organ failure, based on strong laboratory evidence and observational studies.12 It is uncertain whether this association is related to hemolysis or other storage-induced lesions, and it has not been mitigated by transfusion of shorter-storage RBCs in randomized trials. Some of the deleterious effects appear to be mitigated by modifications such as leukoreduction3,13 and/or saline washing14,15 or avoided by use of autologous transfusions16 and restrictive transfusion practices.17,18 Pretransfusion washing of stored RBC and platelet units is an accepted and valuable therapeutic option for patients with repeated and/or severe allergic or febrile transfusion reactions. Recent data from randomized and observational clinical trials demonstrate that pretransfusion washing reduces inflammatory and immunologic complications, including recurrence of acute leukemia,15 elevations of interleukin-6 and C-reactive protein after pediatric cardiac surgery,14 transfusion-related acute lung injury, and transfusion-associated circulatory overload.13 However, washing of RBCs may not be completely benign. Increased hemolysis may occur in vitro during the washing process, and this may be particularly important for RBCs of longer storage duration, which was associated with a higher complication rate in post hoc analysis of a randomized trial.14,19,20 Suboptimal platelet function has also been observed in washed platelets.21 Cholette et al14 demonstrated increased morbidity in children following cardiac surgery with cardiopulmonary bypass who received washed RBCs that were previously stored for more than 28 days compared to less than 14 days. This important finding suggests that the benefit of washed RBC transfusions may vary depending on the storage duration prior to washing. Current washing techniques and dilution of RBCs and platelets for transfusion employ normal saline (NS; 0.9% sodium chloride, NaCl). Data analysis of transfusions at our medical center in 2016 reveal that we transfused about 4,600 platelet doses, of which 27% were NS-washed platelets, and about 15,000 RBC, of which 12% were NS-washed RBCs. Our washing protocol is indicated for patients with documented IgA deficiency, history of severe allergic transfusion reaction, hematologic malignancies, intrauterine transfusions, newborns, neonatal alloimmune thrombocytopenic purpura, priming the extracorporeal membrane oxygenation circuit for infants younger than 1 year of age, neonates with positive circulating maternal isoagglutinin (anti-A/B) testing, patients expressing polyagglutination, liver transplant (two RBC units for initial cooler), pediatric cardiac surgery cases on cardiopulmonary bypass and 5 years old or younger (first RBC unit washed), and for intrauterine and neonatal exchange transfusions. We evaluated whether washing cellular blood components with a more physiologic solution, Plasma-Lyte A (PL-A), might cause less damage to RBCs and better preserve platelet function as compared with NS.22,23 Materials and Methods Washing Solution Compositions Normal Saline NS is an isotonic 0.9% NaCl solution that is composed of 154 mEq/L Na+ and 154 mEq/L Cl– (354.2 mg/dL Na+ and 546.7 mg/dL Cl–) with a neutral pH of 7. However, data showed that the actual pH of NS preparations for infusion is acidic (pH of 5.5-6) at ambient temperature. The acidity of NS is claimed to be due to the plastic pouch container, which is typically composed of polyvinylchloride.24 Plasma-Lyte A PL-A (Baxter Healthcare Corporation, Deerfield, IL) is an isotonic solution containing 140 mEq/L Na+, 5 mEq/L K+, 3 mEq/L Mg+2, 98 mEq/L Cl–, 27 mEq/L sodium acetate trihydrate (C2H3NaO2-3H2O), and 23 mEq/L sodium gluconate (C6H11NaO7). The pH is adjusted with sodium hydroxide to 7.4 (range of 6.5 to 8.0) Table 1.25 Table 1 Characteristics of Normal Saline and Plasma-Lyte A Washing Solutions   Normal Saline  Plasma-Lyte A  Sodium (mEq/L)  154  140  Potassium (mEq/L)  0  5  Calcium (mEq/L)  0  0  Magnesium (mEq/L)  0  3  Chloride (mEq/L)  154  98  Lactate (mEq/L)  0  0  Gluconate (mEq/L)  0  23  Acetate (mEq/L)  0  27  Osmolarity (mOsm/L)  308  294  pH  5.5  7.4    Normal Saline  Plasma-Lyte A  Sodium (mEq/L)  154  140  Potassium (mEq/L)  0  5  Calcium (mEq/L)  0  0  Magnesium (mEq/L)  0  3  Chloride (mEq/L)  154  98  Lactate (mEq/L)  0  0  Gluconate (mEq/L)  0  23  Acetate (mEq/L)  0  27  Osmolarity (mOsm/L)  308  294  pH  5.5  7.4  View Large Table 1 Characteristics of Normal Saline and Plasma-Lyte A Washing Solutions   Normal Saline  Plasma-Lyte A  Sodium (mEq/L)  154  140  Potassium (mEq/L)  0  5  Calcium (mEq/L)  0  0  Magnesium (mEq/L)  0  3  Chloride (mEq/L)  154  98  Lactate (mEq/L)  0  0  Gluconate (mEq/L)  0  23  Acetate (mEq/L)  0  27  Osmolarity (mOsm/L)  308  294  pH  5.5  7.4    Normal Saline  Plasma-Lyte A  Sodium (mEq/L)  154  140  Potassium (mEq/L)  0  5  Calcium (mEq/L)  0  0  Magnesium (mEq/L)  0  3  Chloride (mEq/L)  154  98  Lactate (mEq/L)  0  0  Gluconate (mEq/L)  0  23  Acetate (mEq/L)  0  27  Osmolarity (mOsm/L)  308  294  pH  5.5  7.4  View Large Washed Red Blood Cells RBC units (n = 14) were procured randomly from the blood bank at Strong Memorial Hospital/University of Rochester Medical Center. A prewash sample (12 mL) was collected from each RBC unit as a baseline using a syringe and needle-free sampling site coupler. The prewash hemoglobin and hematocrit were tested using a Sysmex KX-21N cell counter (Sysmex America, Mundelein, IL). Each RBC unit (10 AS-1, 3 AS-3, and 1 CPD-A1) was divided into two equal parts (150 ± 10 mL) using aseptic technique into a 600-mL Blood Transfer Unit Pack with piercing pin (Charter Medical, Winston-Salem, NC). Each half RBC unit was then washed employing a routine clinical protocol on a COBE 2991 Blood Cell Processor (Terumo, Lakewood, CO)26 with 1 L of either NS or PL-A as a wash solution. After the wash solution was added to the RBCs, in an open system method that allows storage for up to 24 hours, a supernatant consisting of the wash solution, plasma proteins, and anticoagulant/preservative was separated from the RBCs by centrifugation. The supernatant was then expressed into a waste bag. The process was repeated for a total of three washing cycles. Once removed from the blood processor, the washed RBCs were resuspended with 20 mL of the wash solution with the syringe and needle-free sampling site coupler. Final volumes and hematocrits for NS and PL-A averaged 87.0 ± 16.4 mL, 61.1%, and 86.7 ± 14.6 mL, 58.8%, respectively. Washed RBCs were maintained at 1°C to 6°C. Samples (12 mL each) were collected from each washed product immediately after washing/resuspending, and at 24, 48, and 72 hours post washing. Cell counts were performed at each time point using a Sysmex KX-21N analyzer. Samples were then immediately centrifuged in a Sorvall RC-3 centrifuge (Beckman Coulter, Carlsbad, CA) for 15 minutes at 4300g. Supernatants were dispensed into labeled cryovials and frozen at –80°C until further testing. Quantification of free heme and free hemoglobin in supernatants was performed using QuantiChrom Assay Kits (Bioassay Systems, Hayward, CA) as previously described.27 Washed Platelets Platelet-rich plasma (PRP) samples were prepared from whole blood and aliquoted (3 mL) from clinical grade platelet concentrates (PC) units (storage period of 5 days) as prepared for transfusion (n = 21). Samples were then washed via an in vitro tube model similar to our standard clinical washing protocol for the COBE 2991.28 Briefly, samples were centrifuged for 10 minutes at 1200g, resuspended in 4.5 mL of either NS or PL-A solution, centrifuged again, and resuspended in 3 mL of the corresponding wash solution (n = 13) or ABO-identical fresh plasma (n = 8). Platelet aggregation studies were performed with light transmission aggregometry on PRP samples (average platelet count 255 ± 27 × 109/L) as per manufacturer’s instructions on a lumiaggregometer (560VS, Chrono-Log Corporation, Havertown, PA) as previously described.29 Adenosine diphosphate (ADP, 10 µmol/L), epinephrine (10 µmol/L), and collagen (2 µg/mL) agonists (Chrono-log Corporation) were added simultaneously to induce platelet aggregation. ADP and epinephrine were added at time 0 followed by collagen at 10 minutes. Platelet aggregations were then quantified over 20 minutes. Washed Platelet Spreading Assay To test the effects of washing and washing fluids on platelet spreading, which is an indication of platelet adherence function, four additional stored PC samples (storage length of 5 days) and four fresh platelet-rich plasma sample controls were washed and spread on fibrinogen-coated coverslips as previously described.29 Briefly, fresh whole blood samples were collected in 3.2% sodium citrate vacutainer tubes from healthy donors who were free of platelet inhibiting medications. Samples were processed immediately and 3 mL of PRP was isolated through centrifugation (10 minutes at 160g). Samples were then washed as described above with either NS or PL-A. Stored PC samples were obtained and washed as described above in both solutions. Platelets were diluted to a final concentration of 33 × 109 platelets/L with respective washing solutions. The platelets were then incubated at 37°C for 45 minutes on glass coverslips that had been coated with 0.25 mg/mL fibrinogen for 2 hours and blocked with 0.5% bovine serum albumin. Unbound platelets were washed away from coverslips with phosphate-buffered saline (PBS) and bound platelets were fixed with 4% paraformaldehyde for 15 minutes and again washed with PBS before being mounted onto glass slides. Platelet spreading was visualized by light microscopy (Zeiss Axiovert 200, Carl Zeiss, Thornwood, NY) and three or four fields were photographed. Platelets were counted and categorized based on their level of spreading by a single technician blinded to the washing solution. Platelets were categorized as (1) not spread if they lacked filopodia, (2) partially spread if they had filopodia but lacked lamellipodia, and (3) fully spread if they demonstrated lamellipodia.29 Statistical Analysis Data are reported as means ± standard error of the mean (SEM) in RBC experiments. Comparisons were performed between RBCs washed with NS vs PL-A using Wilcoxon signed-rank test. Data of the free hemoglobin concentrations of shorter- and longer-storage RBC washed with either NS or PL-A are reported as means ± standard deviation (SD) and the two-tailed Student t tests is used for comparisons. Means ± SD is also used in platelet experiments. Platelet aggregation of platelets washed in NS and PL-A were compared for resuspension with either the washing solution or ABO-identical plasma using two-tailed Student t tests. t tests were also used for platelet spreading experiments where fresh platelet were added to both washing solutions for direct comparisons. A P value of ≤.05 was considered statistically significant. Results Washing With Plasma-Lyte A Leads to Reduced Hemolysis Compared With Normal Saline The median storage age of RBC units was 19 days (range, 10-39 days). The mean ± SEM prewashed hemoglobin and hematocrit were 19.3 ± 0.41 mg/dL and 58.8% ± 1.02%, respectively. Immediately before and after washing, RBC supernatants from both arms had similar mean free hemoglobin and heme Figure 1. Washing reduced both free hemoglobin and free heme by about 37% in NS and 43% in PL-A group, respectively: free hemoglobin (mg/dL) prewash 83.7 ± 15, NS postwash 54 ± 3.5, PL-A postwash 48 ± 3.7 (P = .07); free heme (µmol/L) prewash 46 ± 8.4, NS postwash 29 ± 1.7, PL-A postwash 26 ± 2.2 (P = .28) (Figure 1). Figure 1 View largeDownload slide Free hemoglobin and heme concentrations in washed RBC. Changes in the supernatant concentrations of free hemoglobin (A) and free heme (B) of RBCs washed with 0.9% NaCl (NS) or Plasma-Lyte A (PL-A) over 72 hours. Data are mean ± standard error of the mean. Pre, prewash; Post, postwash. Figure 1 View largeDownload slide Free hemoglobin and heme concentrations in washed RBC. Changes in the supernatant concentrations of free hemoglobin (A) and free heme (B) of RBCs washed with 0.9% NaCl (NS) or Plasma-Lyte A (PL-A) over 72 hours. Data are mean ± standard error of the mean. Pre, prewash; Post, postwash. However, by 24 hours of storage at 1°C to 6°C after washing (the current limit for clinical use in an open system such as the COBE 2991 cell washer), supernatant free hemoglobin and heme levels were significantly increased in RBCs stored in NS as compared to PL-A: free hemoglobin (mg/dL): NS 114 ± 11, PL-A 74 ± 5.7 (109% increase; P < .001); free heme (µmol/L): NS 63 ± 6.1, PL-A 41 ± 3.5 (117% increase; P < .001). These differences between use of NS and PL-A increased over storage time, indicating that prolonged storage after NS washing predisposed to greater hemolysis than PL-A washing (Figure 1). Plasma-Lyte A Mitigates Hemolysis After Washing of Longer Storage Duration RBCs Substantial differences were detected in the free hemoglobin and free heme levels between the shorter-storage RBC units (n = 5; mean storage duration, 14.2 ± 3 days) and longer-storage RBC units (n = 4; mean storage duration, 34 ± 3.8 days, P < .001). Although not statistically significant, the average baseline (before washing) levels of free hemoglobin and free heme were 86% and 99% higher in the longer-storage RBC units than the shorter-storage RBC (free hemoglobin 132 mg/dL vs 71 mg/dL; free heme 75 μmol/L vs 38 μmol/L, respectively) Table 2 and Table 3. No significant changes in the free heme were observed between the shorter-storage and longer-storage RBC units immediately after washing with either NS or PL-A (Table 3). However, a significant increase (P = .025) in the free hemoglobin was observed in the NS-washed longer-storage RBC (Table 2). Substantial increases were seen in the 24-hour free hemoglobin and free heme in the NS-washed longer-storage RBC (43% and 58%, respectively). In the PL-A-washed RBC, in contrast, the 24-hour free hemoglobin and free heme levels were only slightly higher than baseline in the longer-storage RBC (11% and 15%, respectively). These increases in hemolysis were significantly smaller than NS-washed longer-storage RBC units (P = .024 and .012, respectively). Similar patterns were seen in the 48- and 72-hour RBC (Tables 2 and 3). Table 2 Free Hemoglobin Concentrations of Shorter- and Longer-Storage RBC Washed With Normal Saline (NS) or Plasma-Lyte A (PL-A)a Washing Solution  Free Hemoglobin  Shorter Storage RBC  Longer Storage RBC  NS  PL-A  P Valueb  NS  PL-A  P Valueb  Baseline  71 ± 56  —  132 ± 71  —  Immediately after washing  59 ± 19  53 ± 19  .608  55 ± 1.7  40 ± 8  .025  24 hours after washing  85 ± 47  65 ± 18  .418  121 ± 26  72 ± 14  .024  48 hours after washing  101 ± 49  65 ± 21  .186  121 ± 34  94 ± 28  .342  72 hours after washing  137 ± 40  90 ± 18  .055  220 ± 80  142 ± 39  .148  Washing Solution  Free Hemoglobin  Shorter Storage RBC  Longer Storage RBC  NS  PL-A  P Valueb  NS  PL-A  P Valueb  Baseline  71 ± 56  —  132 ± 71  —  Immediately after washing  59 ± 19  53 ± 19  .608  55 ± 1.7  40 ± 8  .025  24 hours after washing  85 ± 47  65 ± 18  .418  121 ± 26  72 ± 14  .024  48 hours after washing  101 ± 49  65 ± 21  .186  121 ± 34  94 ± 28  .342  72 hours after washing  137 ± 40  90 ± 18  .055  220 ± 80  142 ± 39  .148  aData are mean ± standard deviation. bBy the Student t test. View Large Table 2 Free Hemoglobin Concentrations of Shorter- and Longer-Storage RBC Washed With Normal Saline (NS) or Plasma-Lyte A (PL-A)a Washing Solution  Free Hemoglobin  Shorter Storage RBC  Longer Storage RBC  NS  PL-A  P Valueb  NS  PL-A  P Valueb  Baseline  71 ± 56  —  132 ± 71  —  Immediately after washing  59 ± 19  53 ± 19  .608  55 ± 1.7  40 ± 8  .025  24 hours after washing  85 ± 47  65 ± 18  .418  121 ± 26  72 ± 14  .024  48 hours after washing  101 ± 49  65 ± 21  .186  121 ± 34  94 ± 28  .342  72 hours after washing  137 ± 40  90 ± 18  .055  220 ± 80  142 ± 39  .148  Washing Solution  Free Hemoglobin  Shorter Storage RBC  Longer Storage RBC  NS  PL-A  P Valueb  NS  PL-A  P Valueb  Baseline  71 ± 56  —  132 ± 71  —  Immediately after washing  59 ± 19  53 ± 19  .608  55 ± 1.7  40 ± 8  .025  24 hours after washing  85 ± 47  65 ± 18  .418  121 ± 26  72 ± 14  .024  48 hours after washing  101 ± 49  65 ± 21  .186  121 ± 34  94 ± 28  .342  72 hours after washing  137 ± 40  90 ± 18  .055  220 ± 80  142 ± 39  .148  aData are mean ± standard deviation. bBy the Student t test. View Large Table 3 Free Heme Concentrations of Shorter- and Longer-Storage RBC Washed With Either Normal Saline (NS) or Plasma-Lyte A (PL-A)a Washing Solution  Free Heme  Shorter Storage RBC  Longer Storage RBC  NS  PL-A  P Valueb  NS  PL-A  P Valueb  Baseline  38 ± 30  —  75 ± 39  —  Immediately after washing  29 ± 7  28 ± 12  .852  30 ± 6  22 ± 5  .087  24 hours after washing  44 ± 25  35 ± 12  .478  70 ± 13  40 ± 6  .012  48 hours after washing  54 ± 21  37 ± 12  .182  64 ± 9  51 ± 9  .147  72 hours after washing  81 ± 19  48 ± 11  .015  157 ± 40  80 ± 10  .026  Washing Solution  Free Heme  Shorter Storage RBC  Longer Storage RBC  NS  PL-A  P Valueb  NS  PL-A  P Valueb  Baseline  38 ± 30  —  75 ± 39  —  Immediately after washing  29 ± 7  28 ± 12  .852  30 ± 6  22 ± 5  .087  24 hours after washing  44 ± 25  35 ± 12  .478  70 ± 13  40 ± 6  .012  48 hours after washing  54 ± 21  37 ± 12  .182  64 ± 9  51 ± 9  .147  72 hours after washing  81 ± 19  48 ± 11  .015  157 ± 40  80 ± 10  .026  aData are mean ± standard deviation. bBy the Student t test. View Large Table 3 Free Heme Concentrations of Shorter- and Longer-Storage RBC Washed With Either Normal Saline (NS) or Plasma-Lyte A (PL-A)a Washing Solution  Free Heme  Shorter Storage RBC  Longer Storage RBC  NS  PL-A  P Valueb  NS  PL-A  P Valueb  Baseline  38 ± 30  —  75 ± 39  —  Immediately after washing  29 ± 7  28 ± 12  .852  30 ± 6  22 ± 5  .087  24 hours after washing  44 ± 25  35 ± 12  .478  70 ± 13  40 ± 6  .012  48 hours after washing  54 ± 21  37 ± 12  .182  64 ± 9  51 ± 9  .147  72 hours after washing  81 ± 19  48 ± 11  .015  157 ± 40  80 ± 10  .026  Washing Solution  Free Heme  Shorter Storage RBC  Longer Storage RBC  NS  PL-A  P Valueb  NS  PL-A  P Valueb  Baseline  38 ± 30  —  75 ± 39  —  Immediately after washing  29 ± 7  28 ± 12  .852  30 ± 6  22 ± 5  .087  24 hours after washing  44 ± 25  35 ± 12  .478  70 ± 13  40 ± 6  .012  48 hours after washing  54 ± 21  37 ± 12  .182  64 ± 9  51 ± 9  .147  72 hours after washing  81 ± 19  48 ± 11  .015  157 ± 40  80 ± 10  .026  aData are mean ± standard deviation. bBy the Student t test. View Large Plasma-Lyte A Preserves Platelet Function as Compared With Saline The baseline platelet count was 1,478 ± 257 × 109/L (mean ± SD). Following washing, the platelet count dropped by about 5% in both groups (1,413 ± 185 × 109/L; P > .05), demonstrating that the type of washing solution had no influence on platelet recovery. As stored platelets exhibit minimal to no platelet aggregation with single agonists,30,31 baseline platelet aggregation of nonwashed stored PCs was performed with a combination of three agonists (ADP, epinephrine, and collagen) and averaged 40 ± 19%. No aggregation was detected in PCs washed and resuspended in NS. In contrast, aggregation of PCs washed and resuspended in PL-A averaged 29 ± 13% (P < .001 in comparison to NS). Resuspension in ABO-identical plasma improved platelet aggregation significantly for PCs washed in NS or PL-A; however, the PL-A arm still showed superior results in comparison to NS washing (P < .05) Table 4. A representative example of PRP platelet aggregation in response to ADP (10 µmol/L) and epinephrine (10 µmol/L) at time 0 and collagen (2 µg/mL) at 10 minutes is shown in Figure 2. While platelet aggregation of two washed PCs with PL-A (traces 3 and 4) were above 50% for both units, washing the same PC units with NS (traces 1 and 2) showed no measurable platelet aggregation (0%) for both units. Table 4 Light Transmission Platelet Aggregation (%) of Platelet Concentrates (PCs) Washed With Normal Saline (NS) or Plasma-Lyte A (PL-A)a   PCs  NS-Washed PCs  PL-A-Washed PCs  P Value  Baseline (n = 21)  40 ± 19  —  —    Resuspension in same wash solution (n = 13)  —  0  29 ± 13  <.001  Resuspension in ABO-identical plasma (n = 8)  —  41 ± 10  52 ± 5  <.05  P value    <.001  <.05      PCs  NS-Washed PCs  PL-A-Washed PCs  P Value  Baseline (n = 21)  40 ± 19  —  —    Resuspension in same wash solution (n = 13)  —  0  29 ± 13  <.001  Resuspension in ABO-identical plasma (n = 8)  —  41 ± 10  52 ± 5  <.05  P value    <.001  <.05    aData are mean ± standard deviation. View Large Table 4 Light Transmission Platelet Aggregation (%) of Platelet Concentrates (PCs) Washed With Normal Saline (NS) or Plasma-Lyte A (PL-A)a   PCs  NS-Washed PCs  PL-A-Washed PCs  P Value  Baseline (n = 21)  40 ± 19  —  —    Resuspension in same wash solution (n = 13)  —  0  29 ± 13  <.001  Resuspension in ABO-identical plasma (n = 8)  —  41 ± 10  52 ± 5  <.05  P value    <.001  <.05      PCs  NS-Washed PCs  PL-A-Washed PCs  P Value  Baseline (n = 21)  40 ± 19  —  —    Resuspension in same wash solution (n = 13)  —  0  29 ± 13  <.001  Resuspension in ABO-identical plasma (n = 8)  —  41 ± 10  52 ± 5  <.05  P value    <.001  <.05    aData are mean ± standard deviation. View Large Figure 2 View largeDownload slide Light transmission platelet aggregation. The percentage of light transmission (0% set at time 0) is shown for two washed platelet concentrates with NS (traces 1 and 2; blue and black, respectively) and PL-A (traces 3 and 4; red and green, respectively) in response to adenosine diphosphate (10 µmol/L) and epinephrine (10 µmol/L) at time 0 (ADP and Epi), and collagen (2 µg/mL) at 10 minutes simultaneously. Arrows show time of applying each agonist. Figure 2 View largeDownload slide Light transmission platelet aggregation. The percentage of light transmission (0% set at time 0) is shown for two washed platelet concentrates with NS (traces 1 and 2; blue and black, respectively) and PL-A (traces 3 and 4; red and green, respectively) in response to adenosine diphosphate (10 µmol/L) and epinephrine (10 µmol/L) at time 0 (ADP and Epi), and collagen (2 µg/mL) at 10 minutes simultaneously. Arrows show time of applying each agonist. Plasma-Lyte A Improves Platelet Spreading as Compared With Normal Saline As shown in Figure 3C, higher percentages of partially and fully spread platelets were seen when PCs were washed with PL-A as compared with NS (P < .001). The highest percentage of platelets adhering to the fibrinogen coverslip but failing to spread were seen in both stored and fresh platelet samples after washing with NS. The highest percentages of fully spread platelets were observed in fresh PRP samples washed with PL-A (P < .001; Figure 3C). Figure 3 View largeDownload slide Platelet spreading assays. A, Differential interference contrast micrographs demonstrate different stages of platelet spreading that were used as criteria for counting and quantification of platelet spreading. Based on the level of spreading, fully spread platelets were identified by the presence of lamellipodia (arrow) (left); partially spread platelets are indicated by the presence of filopodia (arrowhead) but not lamellipodia (center); and platelets that were round and lacked filopodia were considered not spread (right). B, Examples of platelet spreading of fresh platelets washed with 0.9% NaCl (NS) or Plasma-Lyte A (PL-A) and platelet concentrates washed with NS or PL-A. C, Quantification of whole images derived from experimental samples using images from A as a gauge. Data below the bar chart are percentage of total count (mean ± standard deviation). P < .001 in comparison to NS-washed platelet concentrates (*) and NS-washed fresh platelets (**). Figure 3 View largeDownload slide Platelet spreading assays. A, Differential interference contrast micrographs demonstrate different stages of platelet spreading that were used as criteria for counting and quantification of platelet spreading. Based on the level of spreading, fully spread platelets were identified by the presence of lamellipodia (arrow) (left); partially spread platelets are indicated by the presence of filopodia (arrowhead) but not lamellipodia (center); and platelets that were round and lacked filopodia were considered not spread (right). B, Examples of platelet spreading of fresh platelets washed with 0.9% NaCl (NS) or Plasma-Lyte A (PL-A) and platelet concentrates washed with NS or PL-A. C, Quantification of whole images derived from experimental samples using images from A as a gauge. Data below the bar chart are percentage of total count (mean ± standard deviation). P < .001 in comparison to NS-washed platelet concentrates (*) and NS-washed fresh platelets (**). Discussion RBCs stored for 10 to 39 days and then washed with PL-A experience strikingly less hemolysis during short-term postwashing storage compared with those washed and stored in NS. Whether or not similar degrees of hemolysis would occur in vivo after transfusion is not known. Less hemolysis was observed in the PL-A-washed RBCs stored for more than 4 weeks. We speculate this is due to the improved membrane stability of the longer-stored RBCs washed with PL-A. Although 48 to 72-hour storage of washed RBCs is not clinically employed at present in our center, we evaluated these two time points to measure differences between washing solutions at longer storage intervals. Hemolysis after NS as compared with PL-A washing became progressively worse as postwash storage continued. PL-A washing led to improved postwash platelet aggregation and spreading as compared to NS. Whether such differences would occur in vivo is not known, and the clinical implications are yet to be determined. Reducing free hemoglobin and heme may be important in improving outcomes in transfused patients. Circulating free hemoglobin and heme levels have been associated with thrombosis in animal models,32 transfused patients, and patients with sickle cell disease.33,34 NS, as opposed to PL-A, has been linked to increased renal injury and mortality in animal models and patients.35,36 More physiological levels of potassium and sodium were also reported in RBC washed with Plasma-Lyte as compared to NS washed RBC using the same washing device.37 We speculate that hemolysis may be a contributory cause. Our results indicate that washing RBC with PL-A is a superior approach for maintaining RBC integrity and thus potentially safer than NS. These findings support further investigation of whether NS is truly a suitable diluent for RBC transfusion. PL-A washing might also facilitate lengthening the permissible storage period of washed RBCs. In addition to removing most of the metabolic products and mediators that accumulate during storage, the results of our study confirm that washing blood products with PL-A better maintained RBC structural integrity and preserved platelet function as compared to the commonly used washing solution, NS. The remarkable differences in free heme and hemoglobin concentrations detected in the shorter versus longer stored RBCs washed with NS versus PL-A are additional evidence of the protective effect of PL-A on RBC integrity. PL-A also has potential to partially reverse storage-induced platelet dysfunction. This effect was specific to PL-A in both platelet aggregation and platelet spreading assays, because it was not seen when stored platelets were treated with NS (Figure 2 and Figure 3C). Even when washed platelets were resuspended in ABO-identical plasma, platelet function tests were significantly better in the PL-A-washed platelet arm. Of note, PL-A is almost twice as expensive as NS; the additional cost per liter is approximately $1.00. There are, to our knowledge, no other studies examining the effects of saline vs balanced salt solution washing on RBC lysis and platelet function. Our findings are thus novel. These in vitro studies are at present of unknown clinical relevance. Nonetheless, a growing body of data suggest that free hemoglobin, heme, and nontransferrin-bound iron can mediate morbidity and mortality in animal models.38,39 Our new data support the suggestion that further studies are warranted to determine whether PL-A (or similar balanced crystalloid solutions) has clinical outcome benefits compared to NS when used for resuscitation, coadministration with RBCs and platelets, apheresis, and cell washing in patients requiring crystalloid infusion or transfusion. Acknowledgments We thank Ann Casey from Dr Phipps’ laboratory, blood bank/transfusion service staff and supervisors, and hemostasis and thrombosis laboratory staff for their assistance with these studies. References 1. Healthcare Cost and Utilization Project (HCUP). Facts and Figures: Statistics on Hospital-Based Care in the United States, 2009 . Rockville, MD: Agency for Healthcare Research and Quality; 2011. 2. Popovsky MA, Moore SB. Mechanism of transfusion-related acute lung injury. Blood . 1991; 77: 2299. Google Scholar PubMed  3. Tartter PI, Mohandas K, Azar P, et al.   Randomized trial comparing packed red cell blood transfusion with and without leukocyte depletion for gastrointestinal surgery. Am J Surg . 1998; 176: 462- 466. Google Scholar CrossRef Search ADS PubMed  4. Koch CG, Li L, Sessler DI, et al.   Duration of red-cell storage and complications after cardiac surgery. N Engl J Med . 2008; 358: 1229- 1239. Google Scholar CrossRef Search ADS PubMed  5. 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Google Scholar CrossRef Search ADS PubMed  © American Society for Clinical Pathology, 2018. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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American Journal of Clinical PathologyOxford University Press

Published: Jun 6, 2018

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