Neutrophil extracellular traps in stored canine red blood cell units

Abstract

Background: Neutrophil extracellular traps (NETs), webs of DNA and citrullinated histones extruded from activated neutrophils cause transfusion-related acute lung injury. Supernatants of stored red blood cell (RBC) units might promote NETosis in neutrophils from the units or from transfusion recipients. Hypotheses: (1) NETs form during storage of canine RBC, (2) leukoreduction (LR) before storage of RBC reduces NETosis, and (3) supernatant from stored, nonleukoreduced (NLR) RBC units induces NETosis in healthy canine neutrophils modeling transfusion recipients. Animals: Six healthy purpose-bred research dogs were utilized for blood donation. Methods: Prospective controlled study. RBC units were collected from each dog, aseptically divided into 2 equal subunits, 1 of which was leukoreduced, and stored for 42 days. Stored units were sampled biweekly for quantification of NET markers citrullinated histone H3 (Western blot) and cell-free DNA (cfDNA) (DNA dye binding). Unit supernatants were applied ex vivo to canine neutrophils and extracellular DNA release representing NETosis was assessed. Results: Markers of NETs increased during RBC storage (cfDNA P < .0001 and citrullinated H3 P = .0002) and were higher in NLR than LR units (day 42 LR cfDNA 0.34 ± 0.82 ng/mL vs day 42 NLR 1361.07 ± 741.00 ng/mL, P < .0001; day 42 LR citrullinated H3 0.19 ± 0.13 AU vs NLR 0.57 ± 0.34 AU, P = .007). Isolated neutrophils did not form NETs when exposed to stored canine RBC supernatant. Conclusions and Clinical Importance: NETosis occurs in stored canine NLR RBC units, and is attenuated by LR before storage. NETs might be mediators of transfusion reactions.

Effects of leukoreduction on storage lesions in whole blood and blood components of dogs

“…In our study, leukoreduction successfully met the US FDA threshold of 5 × 10 6 WBC/unit 21 and the less than 1 × 10 6 WBC/unit standard of the Council of Europe 22 . Moreover, leukoreduction caused an almost complete platelets' depletion, confirming the reduction greater than 99% in LR blood canine units 23 . In cold‐stored WB, the platelet count declines by 1% to 2% per day, with limited impact on clotting time or on clot strength as measured by thromboelastography.…”

“…22 Moreover, leukoreduction caused an almost complete platelets' depletion, confirming the reduction greater than 99% in LR blood canine units. 23 In cold-stored WB, the platelet count declines by 1% to 2% per day, with limited impact on clotting time or on clot strength as measured by T A B L E 2 Mean levels and SDs of coagulation variables in whole blood at different sampling times (T0, T35) and in fresh frozen plasma at 1 year of storage thromboelastography. Animal studies have confirmed that platelets from stored WB participate in clot formation.…”

Evaluation of selected hematological, biochemical and oxidative stress parameters in stored canine CPDA-1 whole blood

“…Comparison with platelet-deprived blood products would help to explain the source of MDA in CPDA-1-stored whole blood supernatant. The increase in MDA may also be linked to neutrophil activation and ROS generation, with subsequent oxidation of polyunsaturated lipids, as leukocyte activation in stored canine blood products has been described [17,21]. Studies on human blood products also suggest that early MDA formation may be linked to leukocytes or platelets because the MDA concentration in stored human erythrocytes was shown to increase over time, whereas that measured in the supernatant of RBC concentrates was unchanged [39,40].…”

“…Some ex vivo aging processes have been studied in dog blood, with results indicating release of proinflammatory cytokines and microparticles, changes in erythrocyte volume, potassium release and ATP and 2,3-DPG depletion, along with other biochemical alterations [9,[15][16][17][18]. It was also shown that the presence of leukocytes may partially perpetuate several metabolic disarrangements in canine preserved blood [19][20][21]. Nevertheless, it remains unclear how fast redox system imbalances occur and to what extent oxidative stress correlates with other 'storage lesions' .…”

Mechanistic Understanding of Lung Inflammation: Recent Advances and Emerging Techniques

“…The results indicated that NET markers were increased in stored red blood cells, and demonstrated that leukoreduced red blood cells prior to storage, reduced NET formation. 229 Neutrophils play an important role in TRALI pathogenesis. 230 In a neutrophil and platelet-dependent mouse model of TRALI, NETs were present in the lung microvasculature and plasma.…”