Original Study
Journal of Veterinary Emergency and Critical Care 24(3) 2014, pp 259–263 doi: 10.1111/vec.12160
Cytokine concentration in stored canine erythrocyte concentrates Raffaella Corsi, DVM; Maureen A. McMichael, DVM, DACVECC; Stephanie A. Smith, DVM, MS, DACVIM; Mauria O’Brien, DVM, DACVECC; Jennifer Herring, DVM, DACVECC; Thandeka R. Ngwenyama, DVM; Alyssa Galligan, CVT; Alison N. Beloshapka, MS; Ping Deng, MS and Kelly S. Swanson, PhD
Abstract
Objective – To evaluate the effect of leukoreduction (LR) as compared to standard nonleukoreduced (NLR) units on cytokine concentrations in canine erythrocyte concentrates during regular storage time. Design – Randomized, experimental study. Setting – University teaching hospital. Animals – Ten random-source research dogs. Interventions – One unit of whole blood was collected from each dog and randomized to standard processing (NLR, n = 5) or prestorage LR (n = 5). All units were stored at 4◦ C. Samples were aseptically collected from each unit weekly for 5 weeks. On day 35, 1 mL of blood was collected from each unit and submitted for aerobic culture. Measurement and Main Results – An ELISA assay was used to analyze the concentrations of IL-1, IL-8, TNF␣, and IL-10. There were no significant effects of either group or storage time for IL-1, IL-10, or TNF-␣. IL-8 concentration was significantly increased over the storage period in NLR units, and was significantly higher compared to LR units on days 28 and 35. Aerobic culture was negative on all units. Conclusions – This study demonstrated a marked, storage time-dependent accumulation of IL-8 in canine erythrocyte concentrates. Prestorage LR attenuated the accumulation of IL-8. This chemokine may contribute to the proinflammatory effects of transfusion of stored erythrocyte concentrates. (J Vet Emerg Crit Care 2014; 24(3): 259–263) doi: 10.1111/vec.12160 Keywords: dogs, inflammation, storage lesion, transfusion
Introduction
Abbreviations
Blood transfusions are an essential part of the treatment for critically ill human and veterinary patients. Although blood cells are usually evaluated for antigenic compatibility prior to delivery, multiple studies have
FNHTR Non-LR LR pRBC
From the Department of Veterinary Clinical Sciences, College of Veterinary Medicine (Corsi, McMichael, Obrien, Herring, Ngwenyama, Galligan, Swanson), Department of Biochemistry, College of Medicine (Smith), and Department of Animal Sciences, College of Agricultural, Consumer and Environmental Sciences (Swanson), University of Illinois at Urbana-Champaign, Urbana, IL 61802
documented an association between transfusion and adverse events in the recipient.1–5 In people with major trauma, blood transfusion has been independently associated with increased risk of infection, development of respiratory complications, and admission into the intensive care unit.1 Additionally, trauma patients who received blood transfusions within 24 hours of admission had a higher risk of developing systemic inflammatory response syndrome and death.2 Length of storage appears to be associated with a negative impact on the safety of blood transfusions in people.6, 7 Recently, several studies have documented an increased risk of
The study was performed at the University of Illinois—Urbana campus. The authors declare no conflicts of interests. Presented in part at the European College of Veterinary Internal Medicine Congress, Seville, Spain, September 2011. Address correspondence and reprint requests to Dr. Maureen A. McMichael, Department of Veterinary Clinical Sciences, University of Illinois at Urbana-Champaign, 1008 West Hazelwood Dr, Urbana, IL 61802. Email: [email protected] Submitted June 07, 2012; Accepted December 29, 2013. C Veterinary Emergency and Critical Care Society 2014
febrile nonhemolytic transfusion reactions nonleukoreduction leukoreduction packed RBC
259
R. Corsi et al.
adverse effects associated with transfusion of aged RBCs > 14 days compared with fresh RBC transfusion.8, 9 Transfusion reactions may be either immunologic or nonimmunologic in nature. Immunologic reactions include febrile nonhemolytic transfusion reactions (FNHTRs), transfusion-related acute lung injury and acute, and delayed hemolytic reactions. Nonimmunologic reactions include infectious disease transmission, electrolyte disturbances, and endotoxic shock. FNHTRs are reported with a frequency of 1-6.8% in people3 and 3–5% in companion animals.4 Immunologic transfusion reactions in human recipients, in particular FNHTRs and transfusion-related acute lung injury, are thought to be in part related to transfusion of bioactive molecules derived from leukocytes and platelets,5, 10, 11 including proinflammatory cytokines. Cytokines are regulatory polypeptides that are essential modulators of inflammatory responses. Increased cytokine concentrations have been documented in human erythrocyte concentrates12, 13 and concentrations of bioactive molecules have been shown to increase during storage.9, 14, 15 Leukoreduction (LR) is the process of removing leukocytes (± platelets) from collected blood prior to storage of erythrocytes. Removal of the major cytokine producers, WBCs, at blood collection has the potential to significantly decrease cytokine concentrations in the delivered product. Use of LR attenuates the inflammatory response to transfusion in people16–18 and normal dogs.19 LR of canine blood decreases the formation of procoagulant microparticles in stored erythrocyte concentrates, a process that is likely mediated via the effects of bioactive molecules from contaminating leukocytes and platelets.20 Moreover, LR has been shown to decrease the accumulation of cytokines in erythrocyte concentrates during storage of human blood.11, 12, 21–23 The objective of this study was to evaluate LR and nonLR canine erythrocyte concentrates for changes in inflammatory cytokine concentrations during storage. Our hypotheses were: (1) proinflammatory cytokine concentrations would be lower in LR units as compared to nonLR units; and (2) cytokine concentrations would increase during storage in non-LR units, but not in LR units.
Materials and Methods Collection of supernatant samples The study protocol was approved by the University of Illinois Institutional Animal Care and Use Committee. Whole blood units were collected and processed from 10 random-source healthy research dogs as described in detail previously.20 Briefly, 420 g of whole blood was aseptically collected via standard methods into citratephosphate-dextrose adenine solution. Five of the units 260
were subjected to LR using an inline filter systema that removes leukocytes and platelets. Adequate cell depletion (>99% reduction) was confirmed for the 5 LR units. Centrifugation and plasma removal were performed via standard methods. Sterile sampling ports were placed in each unit to allow removal of samples (35 mL each) on days 0, 7, 14, 21, 28, and 35 of storage. Erythrocyte concentrate aliquots were then centrifuged at 1,850 g for 20 min at 4◦ C without braking. The upper 2/3 of each supernatant was carefully removed, and aliquotted for storage at –80◦ C. Following collection of the samples on day 35, a 1 mL aliquot was obtained from each unit and submitted for aerobic culture. Cytokine assays Quantitative ELISA was used to analyze cytokine concentrations in supernatants. A commercially available ELISA kit was used as recommended for detection of IL-1.b Commercially available antibody sets for canine IL-8, IL-10, and TNF-␣c were utilized as recommended by the supplier. The use of plasma samples containing blood banking anticoagulants and additives was validated for the commercial ELISA kit and all 3 antibody sets. Dilutional linearity, interassay, and intraassay variability, cold-spike recovery, and lower limits of detection were determined, and are reported in table 1. Statistical analysis Determination of data distribution was established using the Kolmogorov–Smirnov test. Cytokine concentrations in supernatants from erythrocyte units were compared by two-way repeated measures ANOVA with evaluation for an effect of both group (NLR versus LR) and storage time (0, 7, 14, 21, 28, 35 days). For post-hoc analysis where the ANOVA established statistical significance, a Tukey test was used for pairwise comparisons. A P value of
Journal of Veterinary Emergency and Critical Care 24(3) 2014, pp 259–263 doi: 10.1111/vec.12160
Cytokine concentration in stored canine erythrocyte concentrates Raffaella Corsi, DVM; Maureen A. McMichael, DVM, DACVECC; Stephanie A. Smith, DVM, MS, DACVIM; Mauria O’Brien, DVM, DACVECC; Jennifer Herring, DVM, DACVECC; Thandeka R. Ngwenyama, DVM; Alyssa Galligan, CVT; Alison N. Beloshapka, MS; Ping Deng, MS and Kelly S. Swanson, PhD
Abstract
Objective – To evaluate the effect of leukoreduction (LR) as compared to standard nonleukoreduced (NLR) units on cytokine concentrations in canine erythrocyte concentrates during regular storage time. Design – Randomized, experimental study. Setting – University teaching hospital. Animals – Ten random-source research dogs. Interventions – One unit of whole blood was collected from each dog and randomized to standard processing (NLR, n = 5) or prestorage LR (n = 5). All units were stored at 4◦ C. Samples were aseptically collected from each unit weekly for 5 weeks. On day 35, 1 mL of blood was collected from each unit and submitted for aerobic culture. Measurement and Main Results – An ELISA assay was used to analyze the concentrations of IL-1, IL-8, TNF␣, and IL-10. There were no significant effects of either group or storage time for IL-1, IL-10, or TNF-␣. IL-8 concentration was significantly increased over the storage period in NLR units, and was significantly higher compared to LR units on days 28 and 35. Aerobic culture was negative on all units. Conclusions – This study demonstrated a marked, storage time-dependent accumulation of IL-8 in canine erythrocyte concentrates. Prestorage LR attenuated the accumulation of IL-8. This chemokine may contribute to the proinflammatory effects of transfusion of stored erythrocyte concentrates. (J Vet Emerg Crit Care 2014; 24(3): 259–263) doi: 10.1111/vec.12160 Keywords: dogs, inflammation, storage lesion, transfusion
Introduction
Abbreviations
Blood transfusions are an essential part of the treatment for critically ill human and veterinary patients. Although blood cells are usually evaluated for antigenic compatibility prior to delivery, multiple studies have
FNHTR Non-LR LR pRBC
From the Department of Veterinary Clinical Sciences, College of Veterinary Medicine (Corsi, McMichael, Obrien, Herring, Ngwenyama, Galligan, Swanson), Department of Biochemistry, College of Medicine (Smith), and Department of Animal Sciences, College of Agricultural, Consumer and Environmental Sciences (Swanson), University of Illinois at Urbana-Champaign, Urbana, IL 61802
documented an association between transfusion and adverse events in the recipient.1–5 In people with major trauma, blood transfusion has been independently associated with increased risk of infection, development of respiratory complications, and admission into the intensive care unit.1 Additionally, trauma patients who received blood transfusions within 24 hours of admission had a higher risk of developing systemic inflammatory response syndrome and death.2 Length of storage appears to be associated with a negative impact on the safety of blood transfusions in people.6, 7 Recently, several studies have documented an increased risk of
The study was performed at the University of Illinois—Urbana campus. The authors declare no conflicts of interests. Presented in part at the European College of Veterinary Internal Medicine Congress, Seville, Spain, September 2011. Address correspondence and reprint requests to Dr. Maureen A. McMichael, Department of Veterinary Clinical Sciences, University of Illinois at Urbana-Champaign, 1008 West Hazelwood Dr, Urbana, IL 61802. Email: [email protected] Submitted June 07, 2012; Accepted December 29, 2013. C Veterinary Emergency and Critical Care Society 2014
febrile nonhemolytic transfusion reactions nonleukoreduction leukoreduction packed RBC
259
R. Corsi et al.
adverse effects associated with transfusion of aged RBCs > 14 days compared with fresh RBC transfusion.8, 9 Transfusion reactions may be either immunologic or nonimmunologic in nature. Immunologic reactions include febrile nonhemolytic transfusion reactions (FNHTRs), transfusion-related acute lung injury and acute, and delayed hemolytic reactions. Nonimmunologic reactions include infectious disease transmission, electrolyte disturbances, and endotoxic shock. FNHTRs are reported with a frequency of 1-6.8% in people3 and 3–5% in companion animals.4 Immunologic transfusion reactions in human recipients, in particular FNHTRs and transfusion-related acute lung injury, are thought to be in part related to transfusion of bioactive molecules derived from leukocytes and platelets,5, 10, 11 including proinflammatory cytokines. Cytokines are regulatory polypeptides that are essential modulators of inflammatory responses. Increased cytokine concentrations have been documented in human erythrocyte concentrates12, 13 and concentrations of bioactive molecules have been shown to increase during storage.9, 14, 15 Leukoreduction (LR) is the process of removing leukocytes (± platelets) from collected blood prior to storage of erythrocytes. Removal of the major cytokine producers, WBCs, at blood collection has the potential to significantly decrease cytokine concentrations in the delivered product. Use of LR attenuates the inflammatory response to transfusion in people16–18 and normal dogs.19 LR of canine blood decreases the formation of procoagulant microparticles in stored erythrocyte concentrates, a process that is likely mediated via the effects of bioactive molecules from contaminating leukocytes and platelets.20 Moreover, LR has been shown to decrease the accumulation of cytokines in erythrocyte concentrates during storage of human blood.11, 12, 21–23 The objective of this study was to evaluate LR and nonLR canine erythrocyte concentrates for changes in inflammatory cytokine concentrations during storage. Our hypotheses were: (1) proinflammatory cytokine concentrations would be lower in LR units as compared to nonLR units; and (2) cytokine concentrations would increase during storage in non-LR units, but not in LR units.
Materials and Methods Collection of supernatant samples The study protocol was approved by the University of Illinois Institutional Animal Care and Use Committee. Whole blood units were collected and processed from 10 random-source healthy research dogs as described in detail previously.20 Briefly, 420 g of whole blood was aseptically collected via standard methods into citratephosphate-dextrose adenine solution. Five of the units 260
were subjected to LR using an inline filter systema that removes leukocytes and platelets. Adequate cell depletion (>99% reduction) was confirmed for the 5 LR units. Centrifugation and plasma removal were performed via standard methods. Sterile sampling ports were placed in each unit to allow removal of samples (35 mL each) on days 0, 7, 14, 21, 28, and 35 of storage. Erythrocyte concentrate aliquots were then centrifuged at 1,850 g for 20 min at 4◦ C without braking. The upper 2/3 of each supernatant was carefully removed, and aliquotted for storage at –80◦ C. Following collection of the samples on day 35, a 1 mL aliquot was obtained from each unit and submitted for aerobic culture. Cytokine assays Quantitative ELISA was used to analyze cytokine concentrations in supernatants. A commercially available ELISA kit was used as recommended for detection of IL-1.b Commercially available antibody sets for canine IL-8, IL-10, and TNF-␣c were utilized as recommended by the supplier. The use of plasma samples containing blood banking anticoagulants and additives was validated for the commercial ELISA kit and all 3 antibody sets. Dilutional linearity, interassay, and intraassay variability, cold-spike recovery, and lower limits of detection were determined, and are reported in table 1. Statistical analysis Determination of data distribution was established using the Kolmogorov–Smirnov test. Cytokine concentrations in supernatants from erythrocyte units were compared by two-way repeated measures ANOVA with evaluation for an effect of both group (NLR versus LR) and storage time (0, 7, 14, 21, 28, 35 days). For post-hoc analysis where the ANOVA established statistical significance, a Tukey test was used for pairwise comparisons. A P value of
