TRANSFUSION PRACTICE Coinfusion of dextrose-containing fluids and red blood cells does not adversely affect in vitro red blood cell quality Amy K. Keir,1,2 Adele L. Hansen,3 Jeannie Callum,4,5 Robert P. Jankov,1,6,7 and Jason P. Acker3,8
BACKGROUND: Transfusion guidelines advise against coinfusing red blood cells (RBCs) with solutions other than 0.9% saline. We evaluated the impact of coinfusion with dextrose-containing fluids (DW) on markers of RBC quality. STUDY DESIGN AND METHODS: A pool-and-split design was used to allow conditions to be tested on each pool within 2 hours of irradiation. Three pools at each storage age (5, 14, and 21 days) were created for each phase. In Phase 1, samples were infused through a neonatal transfusion apparatus alone or with treatment solutions: D5W, D10W, D5W/0.2% saline, and 0.9% saline. In Phase 2, samples were incubated alone or in a 1:1 ratio with treatment solutions and tested after 5, 30, and 180 minutes. Hemolysis, supernatant potassium, RBC indices, morphology, and deformability were measured on all samples. RESULTS: In Phase 1, RBCs transfused alone through the apparatus had higher (p < 0.01) hematocrit, total hemoglobin, and supernatant potassium compared to all other groups. No statistical differences were identified between groups for other measured variables. In Phase 2, mean corpuscular volume of all samples containing DW increased with incubation length and were higher (p < 0.01) than RBCs incubated alone or with 0.9% saline after 30 and 180 minutes. RBCs incubated with D5W and D5W/0.2% saline had greater (p < 0.05) hemolysis than RBCs alone after 180 minutes. CONCLUSION: In vitro characteristics of RBCs coinfused with 0.9% saline or D10W were not adversely impacted. When developing clinical studies in neonates, we recommend use of D10W and a transfusion apparatus that minimizes the contact volume of the coinfusate with the RBC.
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T
he practice of coinfusing red blood cells (RBCs) with dextrose-containing fluids (DW) is advised against due to the potential risk of hemolysis.1 The evidence transfusion guidelines base this recommendation on is limited2 and largely reflects the risk of hemolysis or crenation secondary to coinfusion of RBCs with hypotonic or hypertonic solutions, respectively. Despite this current lack of evidence to either support or refute the practice of coinfusion of stored RBCs with DW, there is increasing interest in the potential safety of this practice in the neonatal population.3,4 This is due, in part, to the increased awareness of the association
ABBREVIATIONS: DW = dextrose-containing fluids; NICU = neonatal intensive care unit. From the 1Division of Neonatology, Department of Paediatrics, Faculty of Medicine, the 4Department of Laboratory Medicine and Pathobiology, Faculty of Medicine, and the 7Department of Physiology, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada; the 2School of Paediatrics and Reproductive Health, University of Adelaide, Adelaide, Australia; the 3Centre for Innovation, Canadian Blood Services, Edmonton, Alberta, Canada; 5Transfusion Medicine and Tissue Banks, Sunnybrook Health Sciences Centre, and the 6Physiology and Experimental Medicine Program, Hospital for Sick Children Research Institute, Toronto, Ontario, Canada; and the 8Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada. Address reprint requests to: Amy K. Keir, Division of Neonatology, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada M5G 1X8; e-mail: [email protected]. Supported by Canadian Blood Services, Medical Insurance Group of Australia (MIGA) Doctors In Training Grant, SickKids Research Training Institute (RTC) Trainee Start-Up Fund, and University of Toronto Division of Neonatology Trainee Grant Program. Received for publication November 21, 2013; revision received January 14, 2014, and accepted January 15, 2014. doi: 10.1111/trf.12618 © 2014 AABB TRANSFUSION 2014;54:2068-2076.
RBCS AND DEXTROSE-CONTAINING FLUIDS
between the development of necrotizing enterocolitis, a potentially devastating disease both with high morbidity and mortality,5 and the receipt of a RBC transfusion within the previous 48 hours.6,7 As a consequence, many neonatal intensive care units (NICUs) routinely withhold enteral feeds from preterm infants before, during, and after transfusion, with the aim of reducing the risk of transfusionassociated necrotizing enterocolitis.8 Although the evidence to support this practice is limited,6,9 it is becoming increasingly common.9 Fasting during RBC transfusion can place infants at risk of hypoglycemia and an additional intravenous (IV) point may be needed to infuse DW to avoid this high-risk situation.10 In some infants, gaining vascular access can be difficult and lead to iatrogenic injury. There is also an emerging association between greater number of skin breaks and poorer cognitive and motor outcomes in preterm infants.11,12 Previous studies examining coinfusion in in vitro settings reflecting neonatal transfusion practice3,13 have suggested that the practice does not lead to hemolysis but significant drawbacks in methodology have led to a general lack of acceptance of these conclusions.4 Our study aims to address these methodologic limitations and provide comprehensive in vitro evidence to determine effects of coinfusion of RBCs and DW on markers of RBC integrity.
MATERIALS AND METHODS Blood product collection and preparation Ethics approval was granted by the Canadian Blood Service’s Research Ethics Board before initiation of the study. Whole blood (n = 45) was collected into CPD from healthy donors (CGR6494B, CPD/SAGM Quad OptiPure RC 9SBT 500 mL, Fenwal, Lake Zurich, IL), rapidly cooled to room temperature (18-24°C), and processed with semiautomated separation of components within 24 hours of the stop bleed time.14 After separation, SAGM was added to the RBCs and leukoreduction occurred by filtration. Once processed, all units were stored at 1 to 6°C for 5, 14, or 21 days. The ages of RBCs were chosen to reflect the most commonly used age of RBCs transfused to infants in Canadian NICUs (14.6 ± 8.3 days).15 Before use in the experiments, the RBCs were irradiated (Gammacell 3000 Elan, MDS Nordion, Ottawa, Ontario, Canada) at a dose of 25 Gy. To be able to test all conditions on the same units within 2 hours of irradiation, a pool-and-split design was used. For Phase 1 of the study, 6 RBC units at each storage age (n = 18) were pooled in pairs and divided equally into the original storage bags to make three sets of 2 pooled RBC units at 5, 14, and 21 days of storage. For Phase 2, 9 RBC units at each of the stated ages (n = 27) were pooled in sets of three and then divided equally into the original storage bags to make three sets of 3 pooled RBC units
at 5, 14, and 21 days of storage. For each phase of the study, the previously outlined experimental conditions were tested on each pool.
Sampling Ten milliliters of RBCs for each experimental condition in Phase 1 and 5 mL of RBCs for Phase 2 conditions were sampled from the pooled units with a 60-mL syringe (Becton, Dickinson and Company, Franklin Lakes, NJ) and an 18-gauge needle (Becton, Dickinson and Company) via a sampling-site coupler (Baxter Healthcare Corporation, Deerfield, IL) having passed through a 170- to 260-μm blood filter (Baxter).
Study design Phase 1: determining the impact of the transfusion apparatus on the RBCs Samples from each of the pools (5 days, n = 3; 14 days, n = 3; 21 days, n = 3) were passed through a neonatal transfusion apparatus either alone (control) or in a 1-to-1 ratio with D5W or D10W solution, D5W and 0.2% saline solution or 0.9% saline solution (isotonic saline control; Baxter). A 5-mL sample was collected in a tube at the end of the transfusion apparatus for testing. The syringe pumps (MedFusion 3500, Medex, Inc., Carlsbad, CA) for the RBCs and coinfusion solutions were both programmed at a rate of 15 mL/hr (3000-g infant, a 120 mL/ kg/day infusion rate for the DW and a 15 mL/kg total volume over 3-hr infusion rate for the RBCs). These conditions were selected as they reflected the fastest rates within the clinical range considered by this study. Standard neonatal transfusion apparatus. A standard neonatal transfusion apparatus was used for the experiments in Phase 1. The apparatus consisted of a 24-gauge IV cannula (Becton, Dickinson and Company) connected to a small-bore (“T-piece”) extension set (ICU Medical, Inc., San Clemente, CA) with two needleless connectors (ICU Medical, Inc.) with extension tubing (Microbore tubing, MED-RX, Oakville, Ontario, Canada) attached. Connected to each extension tube was a 60-mL syringe, one containing the RBCs and the other DW or 0.9% saline (excluding when RBCs were transfused alone, when only one syringe and pump were used). The extension tubing, connected to the syringe with the RBCs, was attached to the needleless connecter closest to the cannula. The small-bore extension set held 0.38 mL in volume and this was the only area in which the RBCs and the treatment solution were in contact with each other. However, when coinfusing it took 10 minutes to collect the volume required for testing in the tube at the end of the apparatus; therefore, the samples were in contact with the DW or 0.9% saline for varying times outside the apparatus that may not be representative of a clinical setting. Volume 54, August 2014 TRANSFUSION
2069
KEIR ET AL.
Phase 2: determining the impact of rate and suspension of infusion Phase 2 was used to investigate the effect of incubation time in the apparatus in a controlled setting. The slowest infusion rate considered in our study was 2.5 mL/hr (500-g infant, a 120 mL/kg/day infusion rate for the DW and a 15 mL/kg total volume over 3-hr infusion rate for the RBCs). The DW and RBCs would therefore be in contact in the tubing line for approximately 4 minutes, using the previously described apparatus in which tubing containing the coinfusion solution and RBC mixture holds 0.38 mL. Samples from each of the pools (5 days, n = 3; 14 days, n = 3; 21 days, n = 3) were stored in a tube for 5 minutes to mimic the slowest infusion rates being considered, 30 minutes to simulate the situation of a temporarily suspended transfusion, or 3 hours to simulate a situation where the transfusion was suspended for its entire duration, either as RBCs independently (control) or with RBCs in a 1:1 ratio with D5W or D10W solution, D5W with 0.2% saline solution, or 0.9% saline (isotonic saline control).
RBC in vitro assays All samples were assessed for hemolysis, supernatant potassium levels, morphology, hematologic indices, and deformability. Supernatant and total hemoglobin (Hb), used to calculate percentage hemolysis, were measured using a Drabkin’s-based method,16 and spun hematocrit (Hct) was measured using a Hct centrifuge (Haematokrit, Andreas Hettich GmbH & Co., Tuttlingen, Germany). The Hct of the 0.9% saline control of the same age was used in the hemolysis calculation for all conditions involving DW to limit the impact of RBC swelling on the percent hemolysis calculation. Extracellular K+ concentration was determined using supernatants, obtained by centrifuging a sample at 2200 × g at 4°C for 10 minutes and indirect potentiometry using ion-selective electrode methodology on a cellular analysis system (DXC 800, Beckman Coulter, Inc., Fullerton, CA). The corrected K+ values were calculated by dividing the concentration (mmol/L) by the total Hb concentration g/L measured using the Drabkin’s method to obtain the potassium mmol/g Hb. This was undertaken due to the dilution of the samples that occurred in the coinfused treatment groups. RBC hematologic indices, including mean corpuscular volume (MCV), mean corpuscular Hb, mean corpuscular Hb concentration, Hb, and Hct were determined using an automated cell counter (Coulter AcT, Beckman Coulter, New York, NY). RBC gross morphology was evaluated by microscopic examination (Eclipse TE2000-U, Nikon, Tokyo, Japan) of both fixed and Wright-stained blood smears. Approximately 10 fields of view were visualized by blinded trained reviewers for each smear and 100 cells were graded based on their morphology, as previously described.17 RBC deformability was calculated using a laser-assisted optical 2070
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rotational cell analyzer (Mechatronics, Zwaage, The Netherlands) as previously described.18
Statistical analysis Mean and standard deviation were calculated by treatment groups (coinfusion solution) to describe data distribution for each RBC characteristic. Linear mixed model analyses were performed to test the effects of the solution and storage age on the product quality. In the mixed model, the random effects were estimated for each of the product units to control for the clustering problem. Posthoc tests were implemented to identify groups, which were significantly different from others. Tukey’s method was used to obtain the adjusted p values in the post-hoc tests. The effect of the storage age on the quality was examined within each treatment. For the Phase 2 data, we also explored the effect of incubation time on the product quality. A p value of
BACKGROUND: Transfusion guidelines advise against coinfusing red blood cells (RBCs) with solutions other than 0.9% saline. We evaluated the impact of coinfusion with dextrose-containing fluids (DW) on markers of RBC quality. STUDY DESIGN AND METHODS: A pool-and-split design was used to allow conditions to be tested on each pool within 2 hours of irradiation. Three pools at each storage age (5, 14, and 21 days) were created for each phase. In Phase 1, samples were infused through a neonatal transfusion apparatus alone or with treatment solutions: D5W, D10W, D5W/0.2% saline, and 0.9% saline. In Phase 2, samples were incubated alone or in a 1:1 ratio with treatment solutions and tested after 5, 30, and 180 minutes. Hemolysis, supernatant potassium, RBC indices, morphology, and deformability were measured on all samples. RESULTS: In Phase 1, RBCs transfused alone through the apparatus had higher (p < 0.01) hematocrit, total hemoglobin, and supernatant potassium compared to all other groups. No statistical differences were identified between groups for other measured variables. In Phase 2, mean corpuscular volume of all samples containing DW increased with incubation length and were higher (p < 0.01) than RBCs incubated alone or with 0.9% saline after 30 and 180 minutes. RBCs incubated with D5W and D5W/0.2% saline had greater (p < 0.05) hemolysis than RBCs alone after 180 minutes. CONCLUSION: In vitro characteristics of RBCs coinfused with 0.9% saline or D10W were not adversely impacted. When developing clinical studies in neonates, we recommend use of D10W and a transfusion apparatus that minimizes the contact volume of the coinfusate with the RBC.
2068
TRANSFUSION Volume 54, August 2014
T
he practice of coinfusing red blood cells (RBCs) with dextrose-containing fluids (DW) is advised against due to the potential risk of hemolysis.1 The evidence transfusion guidelines base this recommendation on is limited2 and largely reflects the risk of hemolysis or crenation secondary to coinfusion of RBCs with hypotonic or hypertonic solutions, respectively. Despite this current lack of evidence to either support or refute the practice of coinfusion of stored RBCs with DW, there is increasing interest in the potential safety of this practice in the neonatal population.3,4 This is due, in part, to the increased awareness of the association
ABBREVIATIONS: DW = dextrose-containing fluids; NICU = neonatal intensive care unit. From the 1Division of Neonatology, Department of Paediatrics, Faculty of Medicine, the 4Department of Laboratory Medicine and Pathobiology, Faculty of Medicine, and the 7Department of Physiology, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada; the 2School of Paediatrics and Reproductive Health, University of Adelaide, Adelaide, Australia; the 3Centre for Innovation, Canadian Blood Services, Edmonton, Alberta, Canada; 5Transfusion Medicine and Tissue Banks, Sunnybrook Health Sciences Centre, and the 6Physiology and Experimental Medicine Program, Hospital for Sick Children Research Institute, Toronto, Ontario, Canada; and the 8Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada. Address reprint requests to: Amy K. Keir, Division of Neonatology, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada M5G 1X8; e-mail: [email protected]. Supported by Canadian Blood Services, Medical Insurance Group of Australia (MIGA) Doctors In Training Grant, SickKids Research Training Institute (RTC) Trainee Start-Up Fund, and University of Toronto Division of Neonatology Trainee Grant Program. Received for publication November 21, 2013; revision received January 14, 2014, and accepted January 15, 2014. doi: 10.1111/trf.12618 © 2014 AABB TRANSFUSION 2014;54:2068-2076.
RBCS AND DEXTROSE-CONTAINING FLUIDS
between the development of necrotizing enterocolitis, a potentially devastating disease both with high morbidity and mortality,5 and the receipt of a RBC transfusion within the previous 48 hours.6,7 As a consequence, many neonatal intensive care units (NICUs) routinely withhold enteral feeds from preterm infants before, during, and after transfusion, with the aim of reducing the risk of transfusionassociated necrotizing enterocolitis.8 Although the evidence to support this practice is limited,6,9 it is becoming increasingly common.9 Fasting during RBC transfusion can place infants at risk of hypoglycemia and an additional intravenous (IV) point may be needed to infuse DW to avoid this high-risk situation.10 In some infants, gaining vascular access can be difficult and lead to iatrogenic injury. There is also an emerging association between greater number of skin breaks and poorer cognitive and motor outcomes in preterm infants.11,12 Previous studies examining coinfusion in in vitro settings reflecting neonatal transfusion practice3,13 have suggested that the practice does not lead to hemolysis but significant drawbacks in methodology have led to a general lack of acceptance of these conclusions.4 Our study aims to address these methodologic limitations and provide comprehensive in vitro evidence to determine effects of coinfusion of RBCs and DW on markers of RBC integrity.
MATERIALS AND METHODS Blood product collection and preparation Ethics approval was granted by the Canadian Blood Service’s Research Ethics Board before initiation of the study. Whole blood (n = 45) was collected into CPD from healthy donors (CGR6494B, CPD/SAGM Quad OptiPure RC 9SBT 500 mL, Fenwal, Lake Zurich, IL), rapidly cooled to room temperature (18-24°C), and processed with semiautomated separation of components within 24 hours of the stop bleed time.14 After separation, SAGM was added to the RBCs and leukoreduction occurred by filtration. Once processed, all units were stored at 1 to 6°C for 5, 14, or 21 days. The ages of RBCs were chosen to reflect the most commonly used age of RBCs transfused to infants in Canadian NICUs (14.6 ± 8.3 days).15 Before use in the experiments, the RBCs were irradiated (Gammacell 3000 Elan, MDS Nordion, Ottawa, Ontario, Canada) at a dose of 25 Gy. To be able to test all conditions on the same units within 2 hours of irradiation, a pool-and-split design was used. For Phase 1 of the study, 6 RBC units at each storage age (n = 18) were pooled in pairs and divided equally into the original storage bags to make three sets of 2 pooled RBC units at 5, 14, and 21 days of storage. For Phase 2, 9 RBC units at each of the stated ages (n = 27) were pooled in sets of three and then divided equally into the original storage bags to make three sets of 3 pooled RBC units
at 5, 14, and 21 days of storage. For each phase of the study, the previously outlined experimental conditions were tested on each pool.
Sampling Ten milliliters of RBCs for each experimental condition in Phase 1 and 5 mL of RBCs for Phase 2 conditions were sampled from the pooled units with a 60-mL syringe (Becton, Dickinson and Company, Franklin Lakes, NJ) and an 18-gauge needle (Becton, Dickinson and Company) via a sampling-site coupler (Baxter Healthcare Corporation, Deerfield, IL) having passed through a 170- to 260-μm blood filter (Baxter).
Study design Phase 1: determining the impact of the transfusion apparatus on the RBCs Samples from each of the pools (5 days, n = 3; 14 days, n = 3; 21 days, n = 3) were passed through a neonatal transfusion apparatus either alone (control) or in a 1-to-1 ratio with D5W or D10W solution, D5W and 0.2% saline solution or 0.9% saline solution (isotonic saline control; Baxter). A 5-mL sample was collected in a tube at the end of the transfusion apparatus for testing. The syringe pumps (MedFusion 3500, Medex, Inc., Carlsbad, CA) for the RBCs and coinfusion solutions were both programmed at a rate of 15 mL/hr (3000-g infant, a 120 mL/ kg/day infusion rate for the DW and a 15 mL/kg total volume over 3-hr infusion rate for the RBCs). These conditions were selected as they reflected the fastest rates within the clinical range considered by this study. Standard neonatal transfusion apparatus. A standard neonatal transfusion apparatus was used for the experiments in Phase 1. The apparatus consisted of a 24-gauge IV cannula (Becton, Dickinson and Company) connected to a small-bore (“T-piece”) extension set (ICU Medical, Inc., San Clemente, CA) with two needleless connectors (ICU Medical, Inc.) with extension tubing (Microbore tubing, MED-RX, Oakville, Ontario, Canada) attached. Connected to each extension tube was a 60-mL syringe, one containing the RBCs and the other DW or 0.9% saline (excluding when RBCs were transfused alone, when only one syringe and pump were used). The extension tubing, connected to the syringe with the RBCs, was attached to the needleless connecter closest to the cannula. The small-bore extension set held 0.38 mL in volume and this was the only area in which the RBCs and the treatment solution were in contact with each other. However, when coinfusing it took 10 minutes to collect the volume required for testing in the tube at the end of the apparatus; therefore, the samples were in contact with the DW or 0.9% saline for varying times outside the apparatus that may not be representative of a clinical setting. Volume 54, August 2014 TRANSFUSION
2069
KEIR ET AL.
Phase 2: determining the impact of rate and suspension of infusion Phase 2 was used to investigate the effect of incubation time in the apparatus in a controlled setting. The slowest infusion rate considered in our study was 2.5 mL/hr (500-g infant, a 120 mL/kg/day infusion rate for the DW and a 15 mL/kg total volume over 3-hr infusion rate for the RBCs). The DW and RBCs would therefore be in contact in the tubing line for approximately 4 minutes, using the previously described apparatus in which tubing containing the coinfusion solution and RBC mixture holds 0.38 mL. Samples from each of the pools (5 days, n = 3; 14 days, n = 3; 21 days, n = 3) were stored in a tube for 5 minutes to mimic the slowest infusion rates being considered, 30 minutes to simulate the situation of a temporarily suspended transfusion, or 3 hours to simulate a situation where the transfusion was suspended for its entire duration, either as RBCs independently (control) or with RBCs in a 1:1 ratio with D5W or D10W solution, D5W with 0.2% saline solution, or 0.9% saline (isotonic saline control).
RBC in vitro assays All samples were assessed for hemolysis, supernatant potassium levels, morphology, hematologic indices, and deformability. Supernatant and total hemoglobin (Hb), used to calculate percentage hemolysis, were measured using a Drabkin’s-based method,16 and spun hematocrit (Hct) was measured using a Hct centrifuge (Haematokrit, Andreas Hettich GmbH & Co., Tuttlingen, Germany). The Hct of the 0.9% saline control of the same age was used in the hemolysis calculation for all conditions involving DW to limit the impact of RBC swelling on the percent hemolysis calculation. Extracellular K+ concentration was determined using supernatants, obtained by centrifuging a sample at 2200 × g at 4°C for 10 minutes and indirect potentiometry using ion-selective electrode methodology on a cellular analysis system (DXC 800, Beckman Coulter, Inc., Fullerton, CA). The corrected K+ values were calculated by dividing the concentration (mmol/L) by the total Hb concentration g/L measured using the Drabkin’s method to obtain the potassium mmol/g Hb. This was undertaken due to the dilution of the samples that occurred in the coinfused treatment groups. RBC hematologic indices, including mean corpuscular volume (MCV), mean corpuscular Hb, mean corpuscular Hb concentration, Hb, and Hct were determined using an automated cell counter (Coulter AcT, Beckman Coulter, New York, NY). RBC gross morphology was evaluated by microscopic examination (Eclipse TE2000-U, Nikon, Tokyo, Japan) of both fixed and Wright-stained blood smears. Approximately 10 fields of view were visualized by blinded trained reviewers for each smear and 100 cells were graded based on their morphology, as previously described.17 RBC deformability was calculated using a laser-assisted optical 2070
TRANSFUSION Volume 54, August 2014
rotational cell analyzer (Mechatronics, Zwaage, The Netherlands) as previously described.18
Statistical analysis Mean and standard deviation were calculated by treatment groups (coinfusion solution) to describe data distribution for each RBC characteristic. Linear mixed model analyses were performed to test the effects of the solution and storage age on the product quality. In the mixed model, the random effects were estimated for each of the product units to control for the clustering problem. Posthoc tests were implemented to identify groups, which were significantly different from others. Tukey’s method was used to obtain the adjusted p values in the post-hoc tests. The effect of the storage age on the quality was examined within each treatment. For the Phase 2 data, we also explored the effect of incubation time on the product quality. A p value of
