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Neutrophil extracellular traps were released during intraoperative blood salvage in posterior lumbar surgery
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Original Article
Neutrophil extracellular traps were released during intraoperative blood salvage in posterior lumbar surgery ⁎
Li Zheng, Ming Tian , Ye Zhang, Peng Dong, He Yang Department of Anesthesia, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Neutrophil extracellular traps Intraoperative blood salvage Posterior lumbar surgery
The formation of neutrophil extracellular traps (NETs) has been associated with endothelial damage and severe pulmonary dysfunction. The present study aimed to investigate whether this NETosis occurs during intraoperative blood salvage (IBS), and whether the washing procedures before re-transfusion of autologous blood could remove the NET components, including DNA, histones, and myeloperoxidase. The study was performed using blood samples from 20 patients who underwent posterior lumbar surgery at the Beijing Friendship Hospital. The samples were obtained at three time points/sources: peripheral venous blood prior to surgery (baseline), cell salvage collection reservoir (reservoir), and filtered salvaged blood prior to re-transfusion (pretransfusion); blood salvage was accomplished with a Cell Saver 5 system. The plasma was collected after centrifugation of the blood sample. Then the DNA amount was measured using SYTOX Green labeling; the integrity and length of the DNA were roughly evaluated by agarose gel electrophoresis; and the levels of nucleosomes (DNA and histones) and myeloperoxidase were detected using commercial ELISA kits. Extracellular DNA, nucleosomes, and myeloperoxidase were found higher in the reservoir samples and pre-transfusion samples, as compared to the baseline samples. The DNA was primarily non–fragmented with high molecular weight (> 15 kb). And lower levels of these NET components were observed in pre-transfusion samples, compared with the reservoir samples. In conclusion, DNA, histones, and myeloperoxidase were released during IBS, indicating the NET formation by activated neutrophils. Pre-transfusion processing could reduce the NET components but the levels remained significantly elevated compared to the baseline.
1. Introduction The use of autologous transfusion has become a widely accepted medical practice. The reasons for this rapid growth were initially attributed to the high demand of blood in regional blood-banking centers and hospital blood banks that often exceeded the regular supply. In addition, autologous transfusion eliminates potential morbidity and complications that may be observed in recipients from blood donors (such as acute lung injury, transfusion-transmitted acquired immunodeficiency syndrome, hemolysis and post-transfusion hepatitis) [1]. Autologous transfusion has advantages since it excludes the risk of isoimmunization to foreign red blood cell (RBC) and platelet antigens, and it decreases the risk of exposure to infectious agents [2,3]. Furthermore, the possibility of salvaging a major part of RBCs from the patient during surgery is considered a significant advantage, notably when excessive hemorrhage occurs. As an alternative to allogeneic transfusion, autologous transfusion involves the removal and re-transfusion of blood from the surgical field [1–3].
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Intraoperative blood salvage (IBS) is performed routinely during posterior lumbar surgery and provides readily available fresh RBCs for autologous transfusion. Although salvaged blood has an excellent clinical safety profile [4,5], some patients have developed complications, including disseminated intravascular coagulopathy and increased vascular permeability in the lungs or systemic circulation (anasarca) following infusion of large amounts of washed autologous blood, resulting in salvaged blood syndrome [6–9]. It has been suggested that this complication could be mediated by neutrophils that have been damaged during the salvage process and activated by platelets [10–12]. Upon contact with activated platelets or inflammatory stimuli, neutrophils and white blood cells (WBCs) release depolymerized DNA chains, histone proteins, and neutrophil enzymes. This process results in the formation of neutrophil extracellular traps (NETs) and causes dramatic changes in the cellular morphology [13,14]. The process is termed NETosis and can be broadly in two types: a) conventional suicidal NETosis and b) vital NETosis [15]. Conventional suicidal NETosis is initiated by ligand binding to neutrophil Toll-
Corresponding author. E-mail address: [email protected] (M. Tian).
https://doi.org/10.1016/j.transci.2018.03.003
1473-0502/ © 2018 Published by Elsevier Ltd.
Please cite this article as: Tian, M., Transfusion and Apheresis Science (2018), https://doi.org/10.1016/j.transci.2018.03.003
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like receptors and receptors for IgG-Fc [16], whereas vital NETosis can occur following direct exposure to bacteria and lipopolysaccharide conjugates after induction of NETs via Toll-like receptor-mediated activation of platelets. The Toll-like receptor 4 is involved in the activation of NETs by Gram-negative bacteria, while the Toll-like receptor 2 is involved in the activation of NETs by Gram-positive bacteria [15]. The main difference between the two NETosis processes is the destruction of the outer membrane of the polymorphonuclear cells (PMNs) that occurs in conventional NETosis, while in vital NETosis the PMNs continue to function even to the point of becoming anuclear [15]. The activation of NETosis [15,16] requires chromatin unwinding through the action of reactive oxygen species (ROS) [17], elastase, myeloperoxidase (MPO) [18], and histone hypercitrullination by the enzyme peptidylarginine deiminase 4 [19]. Although NETs protect against infection [14,20], excessively high plasma levels of neutrophil components can cause organ damage, [13] such as transfusion-related acute lung injury (TRALI) [21,22], thrombosis [23,24], and autoimmune diseases [25,26]. It has been suggested that in the case of platelet-induced neutrophil NET production that may occur after autologous transfusion, a certain threshold of lipopolysaccharide concentration has to be exceeded in order for the induction of neutrophils to occur [15]. This finding indicates that neutrophils are safeguarded from the production of NETs in the vasculature, unless lipopolysaccharide concentrations reach very high levels, such as in the case of severe infection that in turn can activate the platelets [15]. NET components have been identified in the plasma of patients with deep vein thrombosis [27], systemic thrombotic microangiopathies [23], autoimmune diseases [28], and TRALI [21,22]. Since the neutrophils are activated to release extracellular DNA and histones during posterior lumbar surgery, the present study aimed to evaluate the effect of IBS on the aforementioned components prior to re-transfusion of autologous blood. Accordingly, three types of blood samples derived from patients that were scheduled to undergo posterior lumbar surgery were analyzed. The presence of NET components was examined, in terms of the plasma levels of extracellular DNA, histone, and MPO obtained from the peripheral venous blood of the patients. The levels of these parameters were compared among the following blood samples: prior to surgery, cell salvage collection reservoir, and washed, centrifuged, and filtered salvaged blood prior to re-transfusion into the patient.
Braintree, MA, USA) that is used routinely at the study site. The device collects shed blood mixed with heparinized saline from the surgical field. The blood is stored in a collection reservoir that contains an integral 150-μm filter to remove debris. Following successful salvage of 1 L of blood a centrifugation is conducted. Packed RBCs accumulate in the centrifuge bowl and are detected using a light sensor. The RBCs are washed with 1 L of saline. The process results in a final product of approximately 225 mL of autologous non-leukoreduced RBC units that are pumped to an infusion bag for re-transfusion to the patient. The whole cycle lasts approximately 3 min. All the cell salvage procedures were conducted by a blinded investigator who was an expert in using the Cell Saver 5 system.
2. Material and methods
The DNA was isolated from plasma using a DNA isolation kit, according to the manufacturer’s instructions (Omega Bio-Tek, Norcross, GA, USA). The DNA was subjected to 1% agarose gel electrophoresis in the presence of ethidium bromide and visualized with a gel scanning system (Bio-Rad, Hercules, CA, USA).
2.4. Blood samples The blood samples were collected at three stages of the procedure: from peripheral veins following anesthesia induction and prior to surgery (baseline); from the blood salvage collection reservoir and prior to debris removal (reservoir); and after washing, centrifugation, filtration, and passage through a standard y-type blood transfusion set (170-μm filter) prior to reinfusion into the patient (pre-transfusion). At each stage, 2 mL of blood were collected into tubes containing EDTA (BD Vacutainer EDTA Tubes, Franklin Lakes, NJ, USA). The blood samples were centrifuged for 10 min at 1500 × g. The plasma was collected and centrifuged for 10 min at 3000 × g. The plasma was stored at −80 °C. 2.5. DNA quantification The plasma was diluted in phosphate-buffered saline (PBS; GIBCO, Invitrogen Inc., Carlsbad, CA, USA). A total of 50 μl of diluted plasma were mixed with an equal volume of PBS containing SYTOX Green (final concentration: 1 μM; Invitrogen Inc., Carlsbad, CA, USA) for DNA labeling [20]. Fluorescence was recorded using a fluorometer (Fluoroskan; Thermo Fisher Scientific, Waltham, MA, USA). The autofluorescence was determined from samples mixed with PBS without SYTOX Green for the background determination. The DNA concentrations were calculated based on a standard DNA concentration curve (Invitrogen Inc., Carlsbad, CA, USA). 2.6. Isolation and visualization of DNA
2.1. Patients and ethical approval The study was approved by the Institutional Review Board of Beijing Friendship Hospital. Twenty patients provided written informed consent for their participation in the study. Eligibility criteria were: 1) adult patients between 18 and 65 years of age; 2) male or female; 3) American Society of Anesthesiologists (ASA) Class I or II; and 4) scheduled to undergo posterior lumbar surgery. The exclusion criteria were: 1) history of myocardial infarction, stroke, infection, trauma, cancer, or autoimmune disease; or 2) allogeneic transfusion.
2.7. Quantification of nucleosomes and MPO The plasma was diluted at a 1:100 ratio in PBS with 0.1% bovine serum albumin. The concentration of nucleosomes was quantified by an enzyme-linked immunosorbent assay (ELISA) kit (Cell Death Detection kit containing antibodies against histones and DNA, Roche, Indianapolis, IN, USA), according to the manufacturer’s instructions. The plasma concentration of MPO was quantified using a commercially available ELISA kit (Zen MPO ELISA, Invitrogen Inc., Carlsbad, CA, USA), according to the manufacturer’s instructions.
2.2. Anesthesia Posterior lumbar surgery was carried out under general anesthesia. All patients received total intravenous anesthesia with propofol (Propolipid, Fresenius Kabi, Bad Homburg vor der Höhe, Germany) and sufentanil (Ultiva, Glaxo-SmithKline, Middlesex, UK). The patients were monitored by electrocardiogram, arterial oxygen saturation, and invasive blood pressure monitoring during the entire operation.
2.8. Western blot analysis for histone H3 The plasma was analyzed by western blotting for histone H3 detection. A total of 10 μl of plasma were mixed with Laemmli buffer supplemented with 5% β-mercaptoethanol (Bio-Rad, Hercules, CA, USA). Following incubation of the samples at 95 °C for 5 min, sodium dodecyl sulfate-polyacrylamide gel (12.5%) electrophoresis was conducted and the proteins were transferred on a polyvinylidene fluoride
2.3. Blood salvage device Blood was salvaged with a Cell Saver 5 system (Harmonetics Corp., 2
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membrane. The membrane was blocked with Tris-buffered saline and Tween 20 containing 5% skim milk, for 1 h at room temperature. Immunoblotting was performed with a primary polyclonal rabbit antibody against histone H3 (Abcam, Cambridge, MA, USA; catalog # ab1791) and horseradish peroxidase-conjugated goat anti-rabbit IgG (Bio-Rad, Hercules, CA, USA) as a secondary antibody. The detection was carried out with a western blotting substrate (Pierce ECL, Thermo Scientific, Rockford, IL, USA). Human recombinant histone H3 (New England Biolabs, Ipswich, MA, USA) was used as a positive control. 2.9. Statistical analysis Fig. 1. The release of high molecular weight DNA during intraoperative blood salvage (IBS) in posterior lumbar surgery. Blood samples were collected at three stages: from peripheral veins following anesthesia induction and prior to surgery (baseline); from the blood salvage collection reservoir and prior to debris removal (reservoir); and from blood following washing, centrifugation, and filtration (pre-transfusion). The levels of extracellular DNA in the blood samples was quantified and visualized. (A) Scatter plots indicating the mean concentration of DNA measured with SYTOX Green nucleic acid staining. The results were expressed as mean ± standard deviation (SD): Baseline, 26.18 ± 24.50 ng/ml; Reservoir, 4996.06 ± 3528.82 ng/ml; Pre-trans, 291.62 ± 266.95 ng/ml. *** P < 0.001 vs. baseline; ### P < 0.001 vs. reservoir. (B) The representative image of agarose gel electrophoresis indicating the detection of DNA based on the molecular weight.
Statistical analysis was carried out using GraphPad Prism (GraphPad software Inc., San Diego, CA, USA). Significant differences were investigated using analysis of variance (ANOVA) of randomized block design and multiple comparisons using the Student-NewmanKeuls post-hoc test. Two-sided P values < 0.05 were considered significant. 3. Results 3.1. Autologous transfusion The blood was collected from the surgical fields of 20 patients undergoing posterior lumbar surgery. The mean amount of perioperative blood loss per patient was 300 mL and none of the patients exhibited any complications related to salvaged blood transfusion. The mean hematocrit (Hct) of peripheral venous blood was 38% before surgery, whereas the mean Hct of the blood collected in the reservoir during surgery was 21%, due to dilution of the blood cells. The mean Hct of the washed blood used for autologous transfusion was 48%. The average duration from the start of the operation until the collection of the subsequent blood sample (when all the surgical procedures accompanied with bleeding had been completed) was 147 (range, 53–255) min. The time between the collection of the second and third blood samples was approximately 5 min. 3.2. High molecular weight DNA was released during IBS in posterior lumbar surgery
Fig. 2. The release of nucleosomes during IBS in posterior lumbar surgery. The levels of nucleosomes were detected in the plasma from the reservoir samples and pre-transfusion samples compared with the baseline samples, using a Cell Death Detection kit containing antibodies against histones and DNA. The results of optical density values were expressed as mean ± SD: Baseline, 0.06 ± 0.04; Reservoir, 3.09 ± 0.32; Pre-trans, 1.65 ± 1.10. *** P < 0.001 vs. baseline; ### P < 0.001 vs. reservoir.
The amounts of extracellular DNA in blood samples were quantified with SYTOX Green nucleic acid staining (Fig. 1A). The DNA amounts in the reservoir samples were increased to a magnitude of 200-fold compared with the DNA amounts of the baseline samples. Following washing, centrifugation, and filtration, the DNA amounts in salvaged blood (pre-transfusion samples) were significantly reduced compared with the levels in the reservoir samples, but the average amount of DNA in the pre-transfusion samples remained 10-fold greater than the baseline. Upon visualizing the DNA in the plasma samples of 20 patients, high molecular weight (> 15 kb) DNA was detected in the reservoir and pre-transfusion samples, indicating that the DNA was primarily non-fragmented. The intensity of the DNA bands derived from the reservoir samples was greater compared with the intensity corresponding to the pre-transfusion samples. No DNA was detected in the baseline samples (Fig. 1B), which was in agreement with the DNA quantification results.
addition, a part of the extracellular nuclear DNA could be removed from the blood during the washing procedure. 3.4. Histone fragments were detectable in salvaged blood The detection of the nucleosomes was confirmed by western blot analysis of histone H3. The histone H3 fragments were detected in the reservoir plasma samples of all patients, and lower in size compared with the native 15-kDa H3 protein (Fig. 3). 3.5. MPO was released during IBS in posterior lumbar surgery The levels of extracellular MPO were increased to a magnitude of 100-fold in the reservoir plasma compared with the baseline samples. Although the amount of MPO was reduced by washing, centrifugation, and filtration, the MPO levels in the pre-transfusion samples remained approximately 10-fold higher compared with the baseline (Fig. 4).
3.3. Salvaged blood samples contain nucleosomes The ELISA assay for histones and DNA indicated that the nucleosome levels were markedly elevated in the reservoir samples compared with the baseline, and were reduced to some extent following salvaged blood processing (Fig. 2). The nuclear and mitochondrial DNA can be distinguished by the presence of histones. Consequently, the data suggested that nuclear DNA was released during posterior lumbar surgery, but do not rule out the presence or absence of mitochondrial DNA. In
4. Discussion The present study strongly suggested the release of DNA, 3
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During IBS, the washing process and exposure of blood to external surfaces can activate neutrophils and platelets, leading to an accumulation of cells on the centrifuge bowl walls. The activated neutrophils and platelets produce large amounts of proteases, proinflammatory mediators, and ROS [40,41]. ROS can induce NETosis, whereas proinflammatory lipids may facilitate NETosis [32]. Substantial numbers of leukocytes may remain in the blood following washing [37,42] and proinflammatory mediator levels have been shown to be elevated in both unwashed [35] and filtered [36,43–45] salvaged blood relative to circulatory levels. In the present study, elevated levels of NET components were observed in reservoir and in pre-transfusion blood samples, which was in agreement with the previous studies [37,42]. Recent research has shown the involvement of NETs in TRALI [21,22]. Large amounts of NETs are found in the microcirculation and alveolar space of lungs after induction of TRALI by autoantibodies. The extracellular histones that are formed by NETs exhibit intense lungdamaging effects that can interfere to a major extent with air gas exchange between the capillary and alveolar compartments [46]. Plasma DNA and histones can stimulate thrombosis and cause cytotoxicity in mice [24,29,47], whereas purified histones transfused into mice can cause platelet aggregation and lung injury [43,48]. Importantly, treatment of mice with DNase1 or antihistone improved the disease, indicating that NETs contribute to TRALI in mice [21,22]. In the present study it was demonstrated that the extracellular DNA, nucleosomes, and MPO levels were significantly higher in reservoir and pre-transfusion samples compared with the preoperative levels. The process of washing and filtering the salvaged blood reduced the NET component levels substantially compared with the reservoir levels, but they remained higher than the preoperative levels. Taken together, the data suggest that NETosis occurs during posterior lumbar surgery and that NETs can be re-transfused into patients. It is conceivable that retransfused NETs might exert toxic [28,48], prothrombotic [23], or immunomodulatory functions [20,25]. This possibility should be studied further. In addition to surgery, NETosis occurs during RBC storage [49]. The levels of nucleosomes and DNA are elevated in non-leukoreduced RBC units, but not in leukoreduced RBC units [49]. Thus, the reduction of the WBCs in the salvaged blood before reinfusion protects against the damage caused by the infusion of NET components, notably in the cases where a large amount of blood is salvaged and re-transfused. In contrast to the aforementioned observations, the reduction of the WBCs increases the cost of autologous transfusion, and the clinical benefit of this process remains controversial [50–53]. At present, most IBS procedures do not include leukofiltration. Our observation of a histone H3 fragment (< 15 kDa) in the reservoir plasma samples of all patients is consistent with a recent report of histone fragmentation in neutrophils in response to cellular activation and NETosis [18]. Furthermore, our reservoir and pre-transfusions samples were found to contain primarily non-fragmented extracellular DNA. Chromosomal DNA derived from apoptotic cells is characteristically fragmented, whereas cleaved DNA is largely absent in necrotic or NETotic WBCs. Thus, the predominance of non-fragmented extracellular DNA in the samples of the present study indicates that it was not derived, at least not mainly, from apoptotic cells. In the present study, although the total amount of released DNA was decreased considerably in the pre-transfusion samples compared with the reservoir samples, such a profound reduction was not noted for the nucleosomes. A possible explanation for this contradictory finding may be attributed to the type of DNA released that may consist of both nuclear and mitochondrial DNA, as opposed to the nucleosomal fraction that contains solely nuclear fragments. Consequently, it may be suggested that the washing procedures can remove a substantial part of the mitochondrial DNA that is not present in the form of nucleosomes, while this process was less efficient in removing nuclear DNA. Consistent with this observation is the presence of the mtDNA damage associated products in transfusion that has been reported by previous
Fig. 3. Western immunoblotting of histone H3 detection in the reservoir plasma samples. The plasma samples were analyzed using SDS-PAGE and probed with the histone H3 antibody. The experiments were carried out in triplicate. A representative image of western blot showed the histone fragments at molecular weights lower than 15 kDa, which is the molecular size of Histone H3.
Fig. 4. MPO was released during IBS in posterior lumbar surgery. The MPO levels were detected using a commercially available ELISA kit in plasma from the reservoir samples and pre-transfusion samples compared to baseline samples. The results were expressed as SD: Baseline, 4.93 ± 2.33 ng/ ml; Reservoir, 451.81 ± 211.04 ng/ml; Pre-trans, 53.97 ± 30.25 ng/ml. ***P < 0.001 vs. baseline; ###P < 0.001 vs. reservoir.
nucleosomes, and MPO during intraoperative blood salvage in patients who underwent posterior lumbar surgery. The levels of the aforementioned NET components were increased in the reservoir samples and the pre-transfusion samples compared with the baseline samples. Compared to the reservoir samples, a mild decrease was noted in the extracellular nucleosome level of the pre-transfusion samples, and substantial reductions with regard to the extracellular DNA and MPO. The data implied activation of neutrophils occurred during IBS. It also suggests that the NET components could be reduced to some extent by the pre-transfusion washing procedures. NETosis is a programed cell death that is distinct from apoptosis and necrosis. During NETosis, neutrophils release DNA and histones in the form of NETs [29]. Both nuclear and mitochondrial DNA can be released to form NETs [14,30]. Several mechanisms have been described in vitro to explain NETosis. Proinflammatory mediators activate the NAPDH oxidase system of neutrophils and potentiate ROS production [31], which, in turn, induces NETosis. Activated neutrophils may deliver NET components by ejecting mitochondrial or nuclear DNA [24,28], chromatin, and granular proteins [17,18]. Although the underlying signaling events of the different mechanisms are not fully understood, NETosis is thought to be an active process that requires neutrophil stimulation [32]. Unwashed blood in the surgical field may be exposed to tissue factors, air, and the inside surfaces of suction systems. Such exposure and stimulation may activate proinflammatory factors, cytokines, and the complement system, all of which can activate neutrophils [33–39]. 4
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studies, thus indicating a pro-inflammatory role of mtDNA and its implication in disorders such as TRALI [54,55]. One the other hand, we supposed the isolation procedures before agarose gel electrophoresis might have caused fragmentation of DNA, during which the nuclear DNA may be more susceptible; hence the result of electrophoresis (Fig. 1B) could not well reflect the authentic level of nuclear DNA. Nevertheless, further experiments will have to be conducted to specifically examine the contribution of mitochondrial DNA in NETosis in these patients.
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5. Conclusion We demonstrated in the present work that, during IBS, DNA, histones, and MPO were released into blood, indicating the NET formation by activated neutrophils. Pre-transfusion processing could reduce the NET components but the level remained significantly elevated compared to the baseline. Future studies should examine whether the presence of NET biomarkers in salvaged blood is predictive of transfusionrelated complications in a larger number of patients, notably patients undergoing procedures associated with greater blood loss in order to evaluate the necessity of leukofiltration. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Authorship contributions Li Zheng and Ming Tian contributed to conception and design; Li Zheng, Ming Tian, Ye Zhang, Peng Dong and He Yang contributed to acquisition of data, or analysis and interpretation of data; Li Zheng, Ming Tian, Ye Zhang, Peng Dong and He Yang have been involved in drafting the manuscript or revising it critically for important intellectual content; all authors have given final approval of the version to be published. Conflict of interest All authors declare that they have no conflict of interest. Acknowledgements We thank the Experimental and Translational Research Center, Beijing Friendship Hospital, Capital Medical University for assistance with the experiments. References [1] Pan JK, Hong KH, Xie H, Luo MH, Guo D, Liu J. The efficacy and safety of autologous blood transfusion drainage in patients undergoing total knee arthroplasty: a meta-analysis of 16 randomized controlled trials. BMC Musculoskelet Disord 2016;17:452. [2] Yan M, Chen G, Fang LL, Liu ZM, Zhang XL. Immunologic changes to autologous transfusion after operational trauma in malignant tumor patients: neopterin and interleukin-2. J Zhejiang Univ Sci B 2005;6:49–52. [3] Pandey P, Chaudhary R, Aggarwal A, Kumar R, Khetan D, Verma A. Transfusionassociated immunomodulation: quantitative changes in cytokines as a measure of immune responsiveness after one time blood transfusion in neurosurgery patients. Asian J Transfus Sci 2010;4:78–85. [4] Yang C, Wang J, Zheng Z, Zhang Z, Liu H, Wang H, et al. Experience of intraoperative cell salvage in surgical correction of spinal deformity: a retrospective review of 124 patients. Medicine (Baltimore) 2016;95:e3339. [5] Han S, Kim G, Ko JS, Sinn DH, Yang JD, Joh JW, et al. Safety of the use of blood salvage and autotransfusion during liver transplantation for hepatocellular carcinoma. Ann Surg 2016;264:339–43. [6] Paparella D, Whitlock R. Safety of salvaged blood and risk of coagulopathy in cardiac surgery. Semin Thromb Hemost 2016;42:166–71. [7] Gu YJ, Vermeijden WJ, de Vries AJ, Hagenaars JA, Graaff R, van Oeveren W. Influence of mechanical cell salvage on red blood cell aggregation, deformability, and 2,3-diphosphoglycerate in patients undergoing cardiac surgery with
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activation and interleukin formation. Vox Sang 2011;100:239–46. [46] Ward PA, Grailer JJ. Acute lung injury and the role of histones. Trans Resp Med 2014;2:1. [47] Brill A, Fuchs TA, Savchenko AS, Thomas GM, Martinod K, Meyer SFD, et al. Neutrophil extracellular traps promote deep vein thrombosis in mice. J Thromb Haemost 2012;10:136–44. [48] Saffarzadeh M, Juenemann C, Queisser MA, Lochnit G, Barreto G, Galuska SP, et al. Neutrophil extracellular traps directly induce epithelial and endothelial cell death: a predominant role of histones. PLoS One 2012;7:e32366–. [49] Fuchs TA, Alvarez JJ, Martinod K, Bhandari AA, Kaufman RM, Wagner DD. Neutrophils release extracellular DNA traps during storage of red blood cell units. Transfusion (Paris) 2013;53:3210–6. [50] Fuchs TA, Bhandari AA, Wagner DD. Histones induce rapid and profound thrombocytopenia in mice. Blood 2011;118:3708–14. [51] Blajchman MA. The clinical benefits of the leukoreduction of blood products. J Trauma 2006;60:83–90. [52] Silliman CC, Moore EE, Johnson JL, Gonzalez RJ, Biffl WL. Transfusion of the injured patient: proceed with caution. Shock 2004;21:291–9. [53] Phelan HA, Sperry JL, Friese RS. Leukoreduction before red blood cell transfusion has no impact on mortality in trauma patients. J Surg Res 2006;130:32–6. [54] Lee YL, King MB, Gonzalez RP, Brevard SB, Frotan MA, Gillespie MN, et al. Blood transfusion products contain mitochondrial DNA damage-associated molecular patterns: a potential effector of transfusion-related acute lung injury. J Surg Res 2014;191:286–9. [55] Lal A, Gomez E, Calloway C. Increased mitochondrial DNA deletions and copy number in transfusion-dependent thalassemia. JCI Insight 2016;1(12). pii: e88150 Aug 4.
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Transfusion and Apheresis Science journal homepage: www.elsevier.com/locate/transci
Original Article
Neutrophil extracellular traps were released during intraoperative blood salvage in posterior lumbar surgery ⁎
Li Zheng, Ming Tian , Ye Zhang, Peng Dong, He Yang Department of Anesthesia, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Neutrophil extracellular traps Intraoperative blood salvage Posterior lumbar surgery
The formation of neutrophil extracellular traps (NETs) has been associated with endothelial damage and severe pulmonary dysfunction. The present study aimed to investigate whether this NETosis occurs during intraoperative blood salvage (IBS), and whether the washing procedures before re-transfusion of autologous blood could remove the NET components, including DNA, histones, and myeloperoxidase. The study was performed using blood samples from 20 patients who underwent posterior lumbar surgery at the Beijing Friendship Hospital. The samples were obtained at three time points/sources: peripheral venous blood prior to surgery (baseline), cell salvage collection reservoir (reservoir), and filtered salvaged blood prior to re-transfusion (pretransfusion); blood salvage was accomplished with a Cell Saver 5 system. The plasma was collected after centrifugation of the blood sample. Then the DNA amount was measured using SYTOX Green labeling; the integrity and length of the DNA were roughly evaluated by agarose gel electrophoresis; and the levels of nucleosomes (DNA and histones) and myeloperoxidase were detected using commercial ELISA kits. Extracellular DNA, nucleosomes, and myeloperoxidase were found higher in the reservoir samples and pre-transfusion samples, as compared to the baseline samples. The DNA was primarily non–fragmented with high molecular weight (> 15 kb). And lower levels of these NET components were observed in pre-transfusion samples, compared with the reservoir samples. In conclusion, DNA, histones, and myeloperoxidase were released during IBS, indicating the NET formation by activated neutrophils. Pre-transfusion processing could reduce the NET components but the levels remained significantly elevated compared to the baseline.
1. Introduction The use of autologous transfusion has become a widely accepted medical practice. The reasons for this rapid growth were initially attributed to the high demand of blood in regional blood-banking centers and hospital blood banks that often exceeded the regular supply. In addition, autologous transfusion eliminates potential morbidity and complications that may be observed in recipients from blood donors (such as acute lung injury, transfusion-transmitted acquired immunodeficiency syndrome, hemolysis and post-transfusion hepatitis) [1]. Autologous transfusion has advantages since it excludes the risk of isoimmunization to foreign red blood cell (RBC) and platelet antigens, and it decreases the risk of exposure to infectious agents [2,3]. Furthermore, the possibility of salvaging a major part of RBCs from the patient during surgery is considered a significant advantage, notably when excessive hemorrhage occurs. As an alternative to allogeneic transfusion, autologous transfusion involves the removal and re-transfusion of blood from the surgical field [1–3].
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Intraoperative blood salvage (IBS) is performed routinely during posterior lumbar surgery and provides readily available fresh RBCs for autologous transfusion. Although salvaged blood has an excellent clinical safety profile [4,5], some patients have developed complications, including disseminated intravascular coagulopathy and increased vascular permeability in the lungs or systemic circulation (anasarca) following infusion of large amounts of washed autologous blood, resulting in salvaged blood syndrome [6–9]. It has been suggested that this complication could be mediated by neutrophils that have been damaged during the salvage process and activated by platelets [10–12]. Upon contact with activated platelets or inflammatory stimuli, neutrophils and white blood cells (WBCs) release depolymerized DNA chains, histone proteins, and neutrophil enzymes. This process results in the formation of neutrophil extracellular traps (NETs) and causes dramatic changes in the cellular morphology [13,14]. The process is termed NETosis and can be broadly in two types: a) conventional suicidal NETosis and b) vital NETosis [15]. Conventional suicidal NETosis is initiated by ligand binding to neutrophil Toll-
Corresponding author. E-mail address: [email protected] (M. Tian).
https://doi.org/10.1016/j.transci.2018.03.003
1473-0502/ © 2018 Published by Elsevier Ltd.
Please cite this article as: Tian, M., Transfusion and Apheresis Science (2018), https://doi.org/10.1016/j.transci.2018.03.003
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like receptors and receptors for IgG-Fc [16], whereas vital NETosis can occur following direct exposure to bacteria and lipopolysaccharide conjugates after induction of NETs via Toll-like receptor-mediated activation of platelets. The Toll-like receptor 4 is involved in the activation of NETs by Gram-negative bacteria, while the Toll-like receptor 2 is involved in the activation of NETs by Gram-positive bacteria [15]. The main difference between the two NETosis processes is the destruction of the outer membrane of the polymorphonuclear cells (PMNs) that occurs in conventional NETosis, while in vital NETosis the PMNs continue to function even to the point of becoming anuclear [15]. The activation of NETosis [15,16] requires chromatin unwinding through the action of reactive oxygen species (ROS) [17], elastase, myeloperoxidase (MPO) [18], and histone hypercitrullination by the enzyme peptidylarginine deiminase 4 [19]. Although NETs protect against infection [14,20], excessively high plasma levels of neutrophil components can cause organ damage, [13] such as transfusion-related acute lung injury (TRALI) [21,22], thrombosis [23,24], and autoimmune diseases [25,26]. It has been suggested that in the case of platelet-induced neutrophil NET production that may occur after autologous transfusion, a certain threshold of lipopolysaccharide concentration has to be exceeded in order for the induction of neutrophils to occur [15]. This finding indicates that neutrophils are safeguarded from the production of NETs in the vasculature, unless lipopolysaccharide concentrations reach very high levels, such as in the case of severe infection that in turn can activate the platelets [15]. NET components have been identified in the plasma of patients with deep vein thrombosis [27], systemic thrombotic microangiopathies [23], autoimmune diseases [28], and TRALI [21,22]. Since the neutrophils are activated to release extracellular DNA and histones during posterior lumbar surgery, the present study aimed to evaluate the effect of IBS on the aforementioned components prior to re-transfusion of autologous blood. Accordingly, three types of blood samples derived from patients that were scheduled to undergo posterior lumbar surgery were analyzed. The presence of NET components was examined, in terms of the plasma levels of extracellular DNA, histone, and MPO obtained from the peripheral venous blood of the patients. The levels of these parameters were compared among the following blood samples: prior to surgery, cell salvage collection reservoir, and washed, centrifuged, and filtered salvaged blood prior to re-transfusion into the patient.
Braintree, MA, USA) that is used routinely at the study site. The device collects shed blood mixed with heparinized saline from the surgical field. The blood is stored in a collection reservoir that contains an integral 150-μm filter to remove debris. Following successful salvage of 1 L of blood a centrifugation is conducted. Packed RBCs accumulate in the centrifuge bowl and are detected using a light sensor. The RBCs are washed with 1 L of saline. The process results in a final product of approximately 225 mL of autologous non-leukoreduced RBC units that are pumped to an infusion bag for re-transfusion to the patient. The whole cycle lasts approximately 3 min. All the cell salvage procedures were conducted by a blinded investigator who was an expert in using the Cell Saver 5 system.
2. Material and methods
The DNA was isolated from plasma using a DNA isolation kit, according to the manufacturer’s instructions (Omega Bio-Tek, Norcross, GA, USA). The DNA was subjected to 1% agarose gel electrophoresis in the presence of ethidium bromide and visualized with a gel scanning system (Bio-Rad, Hercules, CA, USA).
2.4. Blood samples The blood samples were collected at three stages of the procedure: from peripheral veins following anesthesia induction and prior to surgery (baseline); from the blood salvage collection reservoir and prior to debris removal (reservoir); and after washing, centrifugation, filtration, and passage through a standard y-type blood transfusion set (170-μm filter) prior to reinfusion into the patient (pre-transfusion). At each stage, 2 mL of blood were collected into tubes containing EDTA (BD Vacutainer EDTA Tubes, Franklin Lakes, NJ, USA). The blood samples were centrifuged for 10 min at 1500 × g. The plasma was collected and centrifuged for 10 min at 3000 × g. The plasma was stored at −80 °C. 2.5. DNA quantification The plasma was diluted in phosphate-buffered saline (PBS; GIBCO, Invitrogen Inc., Carlsbad, CA, USA). A total of 50 μl of diluted plasma were mixed with an equal volume of PBS containing SYTOX Green (final concentration: 1 μM; Invitrogen Inc., Carlsbad, CA, USA) for DNA labeling [20]. Fluorescence was recorded using a fluorometer (Fluoroskan; Thermo Fisher Scientific, Waltham, MA, USA). The autofluorescence was determined from samples mixed with PBS without SYTOX Green for the background determination. The DNA concentrations were calculated based on a standard DNA concentration curve (Invitrogen Inc., Carlsbad, CA, USA). 2.6. Isolation and visualization of DNA
2.1. Patients and ethical approval The study was approved by the Institutional Review Board of Beijing Friendship Hospital. Twenty patients provided written informed consent for their participation in the study. Eligibility criteria were: 1) adult patients between 18 and 65 years of age; 2) male or female; 3) American Society of Anesthesiologists (ASA) Class I or II; and 4) scheduled to undergo posterior lumbar surgery. The exclusion criteria were: 1) history of myocardial infarction, stroke, infection, trauma, cancer, or autoimmune disease; or 2) allogeneic transfusion.
2.7. Quantification of nucleosomes and MPO The plasma was diluted at a 1:100 ratio in PBS with 0.1% bovine serum albumin. The concentration of nucleosomes was quantified by an enzyme-linked immunosorbent assay (ELISA) kit (Cell Death Detection kit containing antibodies against histones and DNA, Roche, Indianapolis, IN, USA), according to the manufacturer’s instructions. The plasma concentration of MPO was quantified using a commercially available ELISA kit (Zen MPO ELISA, Invitrogen Inc., Carlsbad, CA, USA), according to the manufacturer’s instructions.
2.2. Anesthesia Posterior lumbar surgery was carried out under general anesthesia. All patients received total intravenous anesthesia with propofol (Propolipid, Fresenius Kabi, Bad Homburg vor der Höhe, Germany) and sufentanil (Ultiva, Glaxo-SmithKline, Middlesex, UK). The patients were monitored by electrocardiogram, arterial oxygen saturation, and invasive blood pressure monitoring during the entire operation.
2.8. Western blot analysis for histone H3 The plasma was analyzed by western blotting for histone H3 detection. A total of 10 μl of plasma were mixed with Laemmli buffer supplemented with 5% β-mercaptoethanol (Bio-Rad, Hercules, CA, USA). Following incubation of the samples at 95 °C for 5 min, sodium dodecyl sulfate-polyacrylamide gel (12.5%) electrophoresis was conducted and the proteins were transferred on a polyvinylidene fluoride
2.3. Blood salvage device Blood was salvaged with a Cell Saver 5 system (Harmonetics Corp., 2
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membrane. The membrane was blocked with Tris-buffered saline and Tween 20 containing 5% skim milk, for 1 h at room temperature. Immunoblotting was performed with a primary polyclonal rabbit antibody against histone H3 (Abcam, Cambridge, MA, USA; catalog # ab1791) and horseradish peroxidase-conjugated goat anti-rabbit IgG (Bio-Rad, Hercules, CA, USA) as a secondary antibody. The detection was carried out with a western blotting substrate (Pierce ECL, Thermo Scientific, Rockford, IL, USA). Human recombinant histone H3 (New England Biolabs, Ipswich, MA, USA) was used as a positive control. 2.9. Statistical analysis Fig. 1. The release of high molecular weight DNA during intraoperative blood salvage (IBS) in posterior lumbar surgery. Blood samples were collected at three stages: from peripheral veins following anesthesia induction and prior to surgery (baseline); from the blood salvage collection reservoir and prior to debris removal (reservoir); and from blood following washing, centrifugation, and filtration (pre-transfusion). The levels of extracellular DNA in the blood samples was quantified and visualized. (A) Scatter plots indicating the mean concentration of DNA measured with SYTOX Green nucleic acid staining. The results were expressed as mean ± standard deviation (SD): Baseline, 26.18 ± 24.50 ng/ml; Reservoir, 4996.06 ± 3528.82 ng/ml; Pre-trans, 291.62 ± 266.95 ng/ml. *** P < 0.001 vs. baseline; ### P < 0.001 vs. reservoir. (B) The representative image of agarose gel electrophoresis indicating the detection of DNA based on the molecular weight.
Statistical analysis was carried out using GraphPad Prism (GraphPad software Inc., San Diego, CA, USA). Significant differences were investigated using analysis of variance (ANOVA) of randomized block design and multiple comparisons using the Student-NewmanKeuls post-hoc test. Two-sided P values < 0.05 were considered significant. 3. Results 3.1. Autologous transfusion The blood was collected from the surgical fields of 20 patients undergoing posterior lumbar surgery. The mean amount of perioperative blood loss per patient was 300 mL and none of the patients exhibited any complications related to salvaged blood transfusion. The mean hematocrit (Hct) of peripheral venous blood was 38% before surgery, whereas the mean Hct of the blood collected in the reservoir during surgery was 21%, due to dilution of the blood cells. The mean Hct of the washed blood used for autologous transfusion was 48%. The average duration from the start of the operation until the collection of the subsequent blood sample (when all the surgical procedures accompanied with bleeding had been completed) was 147 (range, 53–255) min. The time between the collection of the second and third blood samples was approximately 5 min. 3.2. High molecular weight DNA was released during IBS in posterior lumbar surgery
Fig. 2. The release of nucleosomes during IBS in posterior lumbar surgery. The levels of nucleosomes were detected in the plasma from the reservoir samples and pre-transfusion samples compared with the baseline samples, using a Cell Death Detection kit containing antibodies against histones and DNA. The results of optical density values were expressed as mean ± SD: Baseline, 0.06 ± 0.04; Reservoir, 3.09 ± 0.32; Pre-trans, 1.65 ± 1.10. *** P < 0.001 vs. baseline; ### P < 0.001 vs. reservoir.
The amounts of extracellular DNA in blood samples were quantified with SYTOX Green nucleic acid staining (Fig. 1A). The DNA amounts in the reservoir samples were increased to a magnitude of 200-fold compared with the DNA amounts of the baseline samples. Following washing, centrifugation, and filtration, the DNA amounts in salvaged blood (pre-transfusion samples) were significantly reduced compared with the levels in the reservoir samples, but the average amount of DNA in the pre-transfusion samples remained 10-fold greater than the baseline. Upon visualizing the DNA in the plasma samples of 20 patients, high molecular weight (> 15 kb) DNA was detected in the reservoir and pre-transfusion samples, indicating that the DNA was primarily non-fragmented. The intensity of the DNA bands derived from the reservoir samples was greater compared with the intensity corresponding to the pre-transfusion samples. No DNA was detected in the baseline samples (Fig. 1B), which was in agreement with the DNA quantification results.
addition, a part of the extracellular nuclear DNA could be removed from the blood during the washing procedure. 3.4. Histone fragments were detectable in salvaged blood The detection of the nucleosomes was confirmed by western blot analysis of histone H3. The histone H3 fragments were detected in the reservoir plasma samples of all patients, and lower in size compared with the native 15-kDa H3 protein (Fig. 3). 3.5. MPO was released during IBS in posterior lumbar surgery The levels of extracellular MPO were increased to a magnitude of 100-fold in the reservoir plasma compared with the baseline samples. Although the amount of MPO was reduced by washing, centrifugation, and filtration, the MPO levels in the pre-transfusion samples remained approximately 10-fold higher compared with the baseline (Fig. 4).
3.3. Salvaged blood samples contain nucleosomes The ELISA assay for histones and DNA indicated that the nucleosome levels were markedly elevated in the reservoir samples compared with the baseline, and were reduced to some extent following salvaged blood processing (Fig. 2). The nuclear and mitochondrial DNA can be distinguished by the presence of histones. Consequently, the data suggested that nuclear DNA was released during posterior lumbar surgery, but do not rule out the presence or absence of mitochondrial DNA. In
4. Discussion The present study strongly suggested the release of DNA, 3
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During IBS, the washing process and exposure of blood to external surfaces can activate neutrophils and platelets, leading to an accumulation of cells on the centrifuge bowl walls. The activated neutrophils and platelets produce large amounts of proteases, proinflammatory mediators, and ROS [40,41]. ROS can induce NETosis, whereas proinflammatory lipids may facilitate NETosis [32]. Substantial numbers of leukocytes may remain in the blood following washing [37,42] and proinflammatory mediator levels have been shown to be elevated in both unwashed [35] and filtered [36,43–45] salvaged blood relative to circulatory levels. In the present study, elevated levels of NET components were observed in reservoir and in pre-transfusion blood samples, which was in agreement with the previous studies [37,42]. Recent research has shown the involvement of NETs in TRALI [21,22]. Large amounts of NETs are found in the microcirculation and alveolar space of lungs after induction of TRALI by autoantibodies. The extracellular histones that are formed by NETs exhibit intense lungdamaging effects that can interfere to a major extent with air gas exchange between the capillary and alveolar compartments [46]. Plasma DNA and histones can stimulate thrombosis and cause cytotoxicity in mice [24,29,47], whereas purified histones transfused into mice can cause platelet aggregation and lung injury [43,48]. Importantly, treatment of mice with DNase1 or antihistone improved the disease, indicating that NETs contribute to TRALI in mice [21,22]. In the present study it was demonstrated that the extracellular DNA, nucleosomes, and MPO levels were significantly higher in reservoir and pre-transfusion samples compared with the preoperative levels. The process of washing and filtering the salvaged blood reduced the NET component levels substantially compared with the reservoir levels, but they remained higher than the preoperative levels. Taken together, the data suggest that NETosis occurs during posterior lumbar surgery and that NETs can be re-transfused into patients. It is conceivable that retransfused NETs might exert toxic [28,48], prothrombotic [23], or immunomodulatory functions [20,25]. This possibility should be studied further. In addition to surgery, NETosis occurs during RBC storage [49]. The levels of nucleosomes and DNA are elevated in non-leukoreduced RBC units, but not in leukoreduced RBC units [49]. Thus, the reduction of the WBCs in the salvaged blood before reinfusion protects against the damage caused by the infusion of NET components, notably in the cases where a large amount of blood is salvaged and re-transfused. In contrast to the aforementioned observations, the reduction of the WBCs increases the cost of autologous transfusion, and the clinical benefit of this process remains controversial [50–53]. At present, most IBS procedures do not include leukofiltration. Our observation of a histone H3 fragment (< 15 kDa) in the reservoir plasma samples of all patients is consistent with a recent report of histone fragmentation in neutrophils in response to cellular activation and NETosis [18]. Furthermore, our reservoir and pre-transfusions samples were found to contain primarily non-fragmented extracellular DNA. Chromosomal DNA derived from apoptotic cells is characteristically fragmented, whereas cleaved DNA is largely absent in necrotic or NETotic WBCs. Thus, the predominance of non-fragmented extracellular DNA in the samples of the present study indicates that it was not derived, at least not mainly, from apoptotic cells. In the present study, although the total amount of released DNA was decreased considerably in the pre-transfusion samples compared with the reservoir samples, such a profound reduction was not noted for the nucleosomes. A possible explanation for this contradictory finding may be attributed to the type of DNA released that may consist of both nuclear and mitochondrial DNA, as opposed to the nucleosomal fraction that contains solely nuclear fragments. Consequently, it may be suggested that the washing procedures can remove a substantial part of the mitochondrial DNA that is not present in the form of nucleosomes, while this process was less efficient in removing nuclear DNA. Consistent with this observation is the presence of the mtDNA damage associated products in transfusion that has been reported by previous
Fig. 3. Western immunoblotting of histone H3 detection in the reservoir plasma samples. The plasma samples were analyzed using SDS-PAGE and probed with the histone H3 antibody. The experiments were carried out in triplicate. A representative image of western blot showed the histone fragments at molecular weights lower than 15 kDa, which is the molecular size of Histone H3.
Fig. 4. MPO was released during IBS in posterior lumbar surgery. The MPO levels were detected using a commercially available ELISA kit in plasma from the reservoir samples and pre-transfusion samples compared to baseline samples. The results were expressed as SD: Baseline, 4.93 ± 2.33 ng/ ml; Reservoir, 451.81 ± 211.04 ng/ml; Pre-trans, 53.97 ± 30.25 ng/ml. ***P < 0.001 vs. baseline; ###P < 0.001 vs. reservoir.
nucleosomes, and MPO during intraoperative blood salvage in patients who underwent posterior lumbar surgery. The levels of the aforementioned NET components were increased in the reservoir samples and the pre-transfusion samples compared with the baseline samples. Compared to the reservoir samples, a mild decrease was noted in the extracellular nucleosome level of the pre-transfusion samples, and substantial reductions with regard to the extracellular DNA and MPO. The data implied activation of neutrophils occurred during IBS. It also suggests that the NET components could be reduced to some extent by the pre-transfusion washing procedures. NETosis is a programed cell death that is distinct from apoptosis and necrosis. During NETosis, neutrophils release DNA and histones in the form of NETs [29]. Both nuclear and mitochondrial DNA can be released to form NETs [14,30]. Several mechanisms have been described in vitro to explain NETosis. Proinflammatory mediators activate the NAPDH oxidase system of neutrophils and potentiate ROS production [31], which, in turn, induces NETosis. Activated neutrophils may deliver NET components by ejecting mitochondrial or nuclear DNA [24,28], chromatin, and granular proteins [17,18]. Although the underlying signaling events of the different mechanisms are not fully understood, NETosis is thought to be an active process that requires neutrophil stimulation [32]. Unwashed blood in the surgical field may be exposed to tissue factors, air, and the inside surfaces of suction systems. Such exposure and stimulation may activate proinflammatory factors, cytokines, and the complement system, all of which can activate neutrophils [33–39]. 4
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studies, thus indicating a pro-inflammatory role of mtDNA and its implication in disorders such as TRALI [54,55]. One the other hand, we supposed the isolation procedures before agarose gel electrophoresis might have caused fragmentation of DNA, during which the nuclear DNA may be more susceptible; hence the result of electrophoresis (Fig. 1B) could not well reflect the authentic level of nuclear DNA. Nevertheless, further experiments will have to be conducted to specifically examine the contribution of mitochondrial DNA in NETosis in these patients.
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5. Conclusion We demonstrated in the present work that, during IBS, DNA, histones, and MPO were released into blood, indicating the NET formation by activated neutrophils. Pre-transfusion processing could reduce the NET components but the level remained significantly elevated compared to the baseline. Future studies should examine whether the presence of NET biomarkers in salvaged blood is predictive of transfusionrelated complications in a larger number of patients, notably patients undergoing procedures associated with greater blood loss in order to evaluate the necessity of leukofiltration. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Authorship contributions Li Zheng and Ming Tian contributed to conception and design; Li Zheng, Ming Tian, Ye Zhang, Peng Dong and He Yang contributed to acquisition of data, or analysis and interpretation of data; Li Zheng, Ming Tian, Ye Zhang, Peng Dong and He Yang have been involved in drafting the manuscript or revising it critically for important intellectual content; all authors have given final approval of the version to be published. Conflict of interest All authors declare that they have no conflict of interest. Acknowledgements We thank the Experimental and Translational Research Center, Beijing Friendship Hospital, Capital Medical University for assistance with the experiments. References [1] Pan JK, Hong KH, Xie H, Luo MH, Guo D, Liu J. The efficacy and safety of autologous blood transfusion drainage in patients undergoing total knee arthroplasty: a meta-analysis of 16 randomized controlled trials. BMC Musculoskelet Disord 2016;17:452. [2] Yan M, Chen G, Fang LL, Liu ZM, Zhang XL. Immunologic changes to autologous transfusion after operational trauma in malignant tumor patients: neopterin and interleukin-2. J Zhejiang Univ Sci B 2005;6:49–52. [3] Pandey P, Chaudhary R, Aggarwal A, Kumar R, Khetan D, Verma A. Transfusionassociated immunomodulation: quantitative changes in cytokines as a measure of immune responsiveness after one time blood transfusion in neurosurgery patients. Asian J Transfus Sci 2010;4:78–85. [4] Yang C, Wang J, Zheng Z, Zhang Z, Liu H, Wang H, et al. Experience of intraoperative cell salvage in surgical correction of spinal deformity: a retrospective review of 124 patients. Medicine (Baltimore) 2016;95:e3339. [5] Han S, Kim G, Ko JS, Sinn DH, Yang JD, Joh JW, et al. Safety of the use of blood salvage and autotransfusion during liver transplantation for hepatocellular carcinoma. Ann Surg 2016;264:339–43. [6] Paparella D, Whitlock R. Safety of salvaged blood and risk of coagulopathy in cardiac surgery. Semin Thromb Hemost 2016;42:166–71. [7] Gu YJ, Vermeijden WJ, de Vries AJ, Hagenaars JA, Graaff R, van Oeveren W. Influence of mechanical cell salvage on red blood cell aggregation, deformability, and 2,3-diphosphoglycerate in patients undergoing cardiac surgery with
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