{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,17]],"date-time":"2026-10-17T11:43:08Z","timestamp":1792237388334,"version":"4.4.0"},"reference-count":55,"publisher":"MDPI AG","issue":"21","license":[{"start":{"date-parts":[[2025,11,3]],"date-time":"2025-11-03T00:00:00Z","timestamp":1762128000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100008666","name":"Fundaci\u00f3 La Marat\u00f3 de TV3","doi-asserted-by":"publisher","award":["20172430"],"award-info":[{"award-number":["20172430"]}],"id":[{"id":"10.13039\/100008666","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100003030","name":"Ag\u00e8ncia de Gesti\u00f3 d'Ajuts Universitaris i de Recerca","doi-asserted-by":"publisher","award":["2021SGR\/00810"],"award-info":[{"award-number":["2021SGR\/00810"]}],"id":[{"id":"10.13039\/501100003030","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cells"],"abstract":"<jats:p>Background: Anemia is frequent in critically ill patients with traumatic brain injury (TBI) and worsens neurological outcomes. Red blood cell (RBC) transfusion is a cornerstone of management, but storage-related biochemical and structural changes may impair oxygen delivery. This study examined the effect of storage duration on osmotic fragility (OF) and free hemoglobin (fHb) in leukodepleted packed RBCs (pRBCs) as indicators of membrane stability and hemolysis. Methods: Twenty-four leukodepleted pRBC units in SAGM (saline, adenine, glucose, and mannitol) solution were analyzed from Day 3 to Day 42. OF was assessed by Beutler\u2019s method with H50 values derived from logistic models, and fHb was quantified spectrophotometrically. Flow cytometry with phosphate-buffered saline (PBS)-induced osmotic stress provided complementary OF data. Results: OF increased significantly beyond 28 days, with Week 6 H50 values exceeding those at Weeks 2 and 4 (p &lt; 0.0001). fHb rose progressively with storage: 7.3 \u00b1 4.3 \u00b5mol\/L (Week 2), 14.6 \u00b1 7.9 (Week 4), and 25.7 \u00b1 12.1 (Week 6) (p &lt; 0.0001). Hemolysis remained below the 0.8% threshold but increased from 0.09% to 0.29% (p &lt; 0.0001). Conclusions: pRBC storage beyond 28 days leads to greater OF and fHb release, reflecting reduced membrane stability. These changes may compromise transfusion efficacy and oxygen delivery in neurocritical care.<\/jats:p>","DOI":"10.3390\/cells14211726","type":"journal-article","created":{"date-parts":[[2025,11,3]],"date-time":"2025-11-03T17:32:01Z","timestamp":1762191121000},"page":"1726","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Osmotic Fragility in Leukodepleted Stored Red Blood Cells: Implications for Neurocritical Care Transfusion Strategies"],"prefix":"10.3390","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0009-0006-0972-6854","authenticated-orcid":false,"given":"Marta","family":"Peris","sequence":"first","affiliation":[{"name":"Neurotraumatology and Neurosurgery Research Unit, Vall d\u2019Hebron Institut de Reserca (VHIR), Vall d\u2019Hebron Barcelona Hospital Campus, Passeig de la Vall d\u2019Hebron 119-129, 08035 Barcelona, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3831-0536","authenticated-orcid":false,"given":"Maria A.","family":"Poca","sequence":"additional","affiliation":[{"name":"Neurotraumatology and Neurosurgery Research Unit, Vall d\u2019Hebron Institut de Reserca (VHIR), Vall d\u2019Hebron Barcelona Hospital Campus, Passeig de la Vall d\u2019Hebron 119-129, 08035 Barcelona, Spain"},{"name":"Department of Neurosurgery, Vall d\u2019Hebron Hospital Universitari, Passeig de la Vall d\u2019Hebron 119-129, 08035 Barcelona, Spain"},{"name":"Department of Surgery (Neurosurgery), Universitat Aut\u00f2noma de Barcelona, 08193 Bellaterra, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1476-8603","authenticated-orcid":false,"given":"Ana","family":"Ortu\u00f1o","sequence":"additional","affiliation":[{"name":"Erythropathology Unit, Service of Hematology, Vall d\u2019Hebron Hospital Universitari, Passeig de la Vall d\u2019Hebron 119-129, 08035 Barcelona, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ver\u00f3nica","family":"Pons","sequence":"additional","affiliation":[{"name":"Blood and Tissue Bank, Vall d\u2019Hebron Hospital Universitari, Passeig de la Vall d\u2019Hebron 119-129, 08035 Barcelona, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nuria","family":"Rodr\u00edguez-Borrero","sequence":"additional","affiliation":[{"name":"Neurotraumatology and Neurosurgery Research Unit, Vall d\u2019Hebron Institut de Reserca (VHIR), Vall d\u2019Hebron Barcelona Hospital Campus, Passeig de la Vall d\u2019Hebron 119-129, 08035 Barcelona, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Desiree","family":"Jurado","sequence":"additional","affiliation":[{"name":"Neurotraumatology Intensive Care Unit, Vall d\u2019Hebron Hospital Universitari, Passeig de la Vall d\u2019Hebron 119-129, 08035 Barcelona, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rafael","family":"Parra-L\u00f3pez","sequence":"additional","affiliation":[{"name":"Blood and Tissue Bank, Vall d\u2019Hebron Hospital Universitari, Passeig de la Vall d\u2019Hebron 119-129, 08035 Barcelona, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marina","family":"Rierola","sequence":"additional","affiliation":[{"name":"Blood and Tissue Bank, Vall d\u2019Hebron Hospital Universitari, Passeig de la Vall d\u2019Hebron 119-129, 08035 Barcelona, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0713-5875","authenticated-orcid":false,"given":"Juan","family":"Sahuquillo","sequence":"additional","affiliation":[{"name":"Neurotraumatology and Neurosurgery Research Unit, Vall d\u2019Hebron Institut de Reserca (VHIR), Vall d\u2019Hebron Barcelona Hospital Campus, Passeig de la Vall d\u2019Hebron 119-129, 08035 Barcelona, Spain"},{"name":"Department of Neurosurgery, Vall d\u2019Hebron Hospital Universitari, Passeig de la Vall d\u2019Hebron 119-129, 08035 Barcelona, Spain"},{"name":"Department of Surgery (Neurosurgery), Universitat Aut\u00f2noma de Barcelona, 08193 Bellaterra, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,11,3]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"3124","DOI":"10.1097\/CCM.0b013e3181b39f1b","article-title":"Clinical practice guideline: Red blood cell transfusion in adult trauma and critical care","volume":"37","author":"Napolitano","year":"2009","journal-title":"Crit. Care Med."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"155","DOI":"10.1089\/neu.2010.1451","article-title":"Anemia in the setting of traumatic brain injury: The arguments for and against liberal transfusion","volume":"28","author":"Utter","year":"2011","journal-title":"J. Neurotrauma"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"82","DOI":"10.1016\/j.wneu.2016.02.076","article-title":"The Negative Impact of Anemia in Outcome from Traumatic Brain Injury","volume":"90","author":"Litofsky","year":"2016","journal-title":"World Neurosurg."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"199","DOI":"10.1186\/s13054-024-04980-6","article-title":"Haemoglobin values, transfusion practices, and long-term outcomes in critically ill patients with traumatic brain injury: A secondary analysis of CENTER-TBI","volume":"28","author":"Guglielmi","year":"2024","journal-title":"Crit. Care"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"185","DOI":"10.1007\/s12028-016-0273-7","article-title":"Association Between Relative Anemia and Early Functional Recovery After Severe Traumatic Brain Injury (TBI)","volume":"25","author":"Carr","year":"2016","journal-title":"Neurocrit. Care"},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"McCullogh, J. (2021). Preparation, Storage, and Characteristics of Whole Blood, Blood Components, and Plasma Derivatives. Transfusion Medicine, Wiley.","DOI":"10.1002\/9781119599586"},{"key":"ref_7","first-page":"27","article-title":"Red blood cell storage lesion: Causes and potential clinical consequences","volume":"17","author":"Yoshida","year":"2019","journal-title":"Blood Transfus."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"6","DOI":"10.1186\/1471-2326-4-6","article-title":"A randomized trial of washed red blood cell and platelet transfusions in adult acute leukemia [ISRCTN76536440]","volume":"4","author":"Blumberg","year":"2004","journal-title":"BMC Hematol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"3","DOI":"10.1111\/tme.12481","article-title":"The administration of blood components: A British Society for Haematology Guideline","volume":"28","author":"Robinson","year":"2018","journal-title":"Transfus. Med."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"3","DOI":"10.1111\/bjh.12747","article-title":"Does prolonged storage of red blood cells cause harm?","volume":"165","author":"Flegel","year":"2014","journal-title":"Br. J. Haematol."},{"key":"ref_11","first-page":"e2952","article-title":"Impact of Storage Lesion on Post-transfusion Rise in Hemoglobin","volume":"10","author":"Sardar","year":"2018","journal-title":"Cureus"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"183","DOI":"10.1159\/000452106","article-title":"Biophysical and Biochemical Markers of Red Blood Cell Fragility","volume":"44","author":"Orbach","year":"2017","journal-title":"Transfus. Med. Hemother."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"107","DOI":"10.3324\/haematol.2011.051789","article-title":"Time-course investigation of SAGM-stored leukocyte-filtered red bood cell concentrates: From metabolism to proteomics","volume":"97","author":"Vaglio","year":"2012","journal-title":"Haematologica"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"e0122655","DOI":"10.1371\/journal.pone.0122655","article-title":"Plasma free hemoglobin and microcirculatory response to fresh or old blood transfusions in sepsis","volume":"10","author":"Damiani","year":"2015","journal-title":"PLoS ONE"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"844","DOI":"10.1111\/j.1537-2995.2011.03100.x","article-title":"Storage lesion: Role of red blood cell breakdown","volume":"51","author":"Lee","year":"2011","journal-title":"Transfusion"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"465","DOI":"10.1161\/CIRCULATIONAHA.110.008698","article-title":"Nitric Oxide Scavenging by Red Blood Cell Microparticles and Cell-Free Hemoglobin as a Mechanism for the Red Cell Storage Lesion","volume":"124","author":"Donadee","year":"2011","journal-title":"Circulation"},{"key":"ref_17","first-page":"82","article-title":"Red blood cell storage: The story so far","volume":"8","author":"Liumbruno","year":"2010","journal-title":"Blood Transfus."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"264","DOI":"10.1016\/S0022-3476(89)80794-2","article-title":"Normal erythrocyte osmotic fragility in hereditary spherocytosis","volume":"114","author":"Korones","year":"1989","journal-title":"J. Pediatr."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1141","DOI":"10.1182\/blood.V59.6.1141.1141","article-title":"The osmotic fragility of erythrocytes after prolonged liquid storage and after reinfusion","volume":"59","author":"Beutler","year":"1982","journal-title":"Blood"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"015001","DOI":"10.1117\/1.BIOS.2.1.015001","article-title":"Hybrid diffuse optical appraisal of peripheral and cerebral changes in critically ill patients receiving red blood cell transfusion","volume":"2","author":"Tagliabue","year":"2025","journal-title":"Biophotonics Discov."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"4529434","DOI":"10.1155\/2016\/4529434","article-title":"Time Dependent Assessment of Morphological Changes: Leukodepleted Packed Red Blood Cells Stored in SAGM","volume":"2016","author":"Mustafa","year":"2016","journal-title":"BioMed Res. Int."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"90","DOI":"10.1111\/j.1365-3148.2012.01139.x","article-title":"Red blood cell storage and cell morphology","volume":"22","author":"Blasi","year":"2012","journal-title":"Transfus. Med."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"175","DOI":"10.3758\/BF03193146","article-title":"G*Power 3: A flexible statistical power analysis program for the social, behavioral, and biomedical sciences","volume":"39","author":"Faul","year":"2007","journal-title":"Behav. Res. Methods"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1149","DOI":"10.3758\/BRM.41.4.1149","article-title":"Statistical power analyses using G*Power 3.1: Tests for correlation and regression analyses","volume":"41","author":"Faul","year":"2009","journal-title":"Behav. Res. Methods"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"66","DOI":"10.3109\/00365515909060410","article-title":"A method for determination of hemoglobin in plasma by near-ultraviolet spectrophotometry","volume":"11","author":"Harboe","year":"1959","journal-title":"Scand. J. Clin. Lab. Investig."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"116","DOI":"10.1111\/j.1423-0410.2009.01249.x","article-title":"A comparative study of common techniques used to measure haemolysis in stored red cell concentrates","volume":"98","author":"Han","year":"2010","journal-title":"Vox Sang."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"e23242","DOI":"10.1002\/jcla.23242","article-title":"Automation of Harboe method for the measurement of plasma free hemoglobin","volume":"34","author":"Chung","year":"2020","journal-title":"J. Clin. Lab. Anal."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"483","DOI":"10.1177\/2211068214532254","article-title":"Flow cytometric analysis of red blood cell osmotic fragility","volume":"19","author":"Yamamoto","year":"2014","journal-title":"J. Lab. Autom."},{"key":"ref_29","unstructured":"Bikos, L.H. (2023). ReCentering Psych Stats, Seattle Pacific University Library."},{"key":"ref_30","unstructured":"Kline, R.B. (2016). Data Preparation and Psychometrics Review. Methodology in the Social Sciences. Principles and Practice of Structural Equation Modeling, The Guilford Press. [5th ed.]."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"e0146021","DOI":"10.1371\/journal.pone.0146021","article-title":"Dose-Response Analysis Using R","volume":"10","author":"Ritz","year":"2016","journal-title":"PLoS ONE"},{"key":"ref_32","unstructured":"R Core Team (2025, July 23). R: A language and environment for statistical computing. Available online: https:\/\/www.bibsonomy.org\/bibtex\/7469ffee3b07f9167cf47e7555041ee7."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1686","DOI":"10.21105\/joss.01686","article-title":"Welcome to the Tidyverse","volume":"4","author":"Wickham","year":"2019","journal-title":"J. Open Source Softw."},{"key":"ref_34","unstructured":"Revelle, W. (2024). Psych: Procedures for Psychological, Psychometric, and Personality Research, Northwestern University."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"78","DOI":"10.1007\/s12028-014-0096-3","article-title":"Hemoglobin Area and Time Index Above 90 g\/L are Associated with Improved 6-Month Functional Outcomes in Patients with Severe Traumatic Brain Injury","volume":"23","author":"Griesdale","year":"2015","journal-title":"Neurocrit. Care"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"439","DOI":"10.1001\/jama.1978.03290050029011","article-title":"Early insults to the injured brain","volume":"240","author":"Miller","year":"1978","journal-title":"JAMA"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"S668","DOI":"10.1097\/01.CCM.0000099348.99451.84","article-title":"Transfusion practice in the critically ill","volume":"31","author":"Corwin","year":"2003","journal-title":"Crit. Care Med."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"89","DOI":"10.1186\/s13054-018-2273-9","article-title":"Transfusion requirements after head trauma: A randomized feasibility controlled trial","volume":"23","author":"Gobatto","year":"2019","journal-title":"Crit. Care"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"409","DOI":"10.1056\/NEJM199902113400601","article-title":"A multicenter, randomized, controlled clinical trial of transfusion requirements in critical care","volume":"340","author":"Hebert","year":"1999","journal-title":"N. Engl. J. Med."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"67","DOI":"10.1227\/NEU.0000000000000575","article-title":"Collaborative European NeuroTrauma Effectiveness Research in Traumatic Brain Injury (CENTER-TBI): A Prospective Longitudinal Observational Study","volume":"76","author":"Maas","year":"2015","journal-title":"Neurosurgery"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"1623","DOI":"10.1001\/jama.2024.20424","article-title":"Restrictive vs Liberal Transfusion Strategy in Patients With Acute Brain Injury: The TRAIN Randomized Clinical Trial","volume":"332","author":"Taccone","year":"2024","journal-title":"JAMA"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"722","DOI":"10.1056\/NEJMoa2404360","article-title":"Liberal or Restrictive Transfusion Strategy in Patients with Traumatic Brain Injury","volume":"391","author":"Turgeon","year":"2024","journal-title":"N. Engl. J. Med."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"1079","DOI":"10.1056\/NEJMoa2410962","article-title":"Liberal or Restrictive Transfusion Strategy in Aneurysmal Subarachnoid Hemorrhage","volume":"392","author":"English","year":"2025","journal-title":"N. Engl. J. Med."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"477","DOI":"10.1016\/j.chest.2024.09.016","article-title":"Red Blood Cell Transfusion in Critically Ill Adults: An American College of Chest Physicians Clinical Practice Guideline","volume":"167","author":"Soghier","year":"2025","journal-title":"Chest"},{"key":"ref_45","first-page":"e314","article-title":"2023 Guideline for the Management of Patients With Aneurysmal Subarachnoid Hemorrhage: A Guideline From the American Heart Association\/American Stroke Association","volume":"54","author":"Hoh","year":"2023","journal-title":"Stroke"},{"key":"ref_46","unstructured":"WHO (2025, August 19). Haemoglobin Concentrations for the Diagnosis of Anaemia and Assessment of Severity. Available online: https:\/\/iris.who.int\/bitstream\/handle\/10665\/85839\/WHO_NMH_NHD_MNM_11.1_eng.pdf."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"1513","DOI":"10.1016\/S1357-2725(02)00087-0","article-title":"Red blood cells","volume":"34","author":"Klinken","year":"2002","journal-title":"Int. J. Biochem. Cell Biol."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"598","DOI":"10.1007\/s12288-016-0769-5","article-title":"Study of Hemolysis During Storage of Blood in the Blood Bank of a Tertiary Health Care Centre","volume":"33","author":"Arif","year":"2017","journal-title":"Indian J. Hematol. Blood Transfus."},{"key":"ref_49","unstructured":"(2025, October 30). European Directorate for the Quality of Medicines & HealthCare. Available online: https:\/\/www.edqm.eu\/en\/blood-guide."},{"key":"ref_50","unstructured":"U.S. Department of Health and Human Services, Food and Drug Administration, & Center for Biologics Evaluation and Research (2025, October 30). Available online: https:\/\/www.fda.gov\/regulatory-information\/search-fda-guidance-documents\/recommendations-development-blood-collection-processing-and-storage-systems-manufacture-blood."},{"key":"ref_51","first-page":"e53417","article-title":"A Case Report of Hemolytic Hyponatremia","volume":"16","year":"2024","journal-title":"Cureus"},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"6","DOI":"10.1006\/jsre.2001.6306","article-title":"Influence of storage on red blood cell rheological properties","volume":"102","author":"Berezina","year":"2002","journal-title":"J. Surg. Res."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"549","DOI":"10.1136\/jcp.2006.037523","article-title":"Assessment of erythrocyte shape by flow cytometry techniques","volume":"60","author":"Piagnerelli","year":"2007","journal-title":"J. Clin. Pathol."},{"key":"ref_54","doi-asserted-by":"crossref","unstructured":"Grigorev, G.V., Lebedev, A.V., Wang, X., Qian, X., Maksimov, G.V., and Lin, L. (2023). Advances in Microfluidics for Single Red Blood Cell Analysis. Biosensors, 13.","DOI":"10.3390\/bios13010117"},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"4065","DOI":"10.1016\/j.jbiomech.2015.10.002","article-title":"Microfluidic deformability analysis of the red cell storage lesion","volume":"48","author":"Matthews","year":"2015","journal-title":"J. Biomech."}],"container-title":["Cells"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2073-4409\/14\/21\/1726\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,11,5]],"date-time":"2025-11-05T05:15:31Z","timestamp":1762319731000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2073-4409\/14\/21\/1726"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,11,3]]},"references-count":55,"journal-issue":{"issue":"21","published-online":{"date-parts":[[2025,11]]}},"alternative-id":["cells14211726"],"URL":"https:\/\/doi.org\/10.3390\/cells14211726","relation":{},"ISSN":["2073-4409"],"issn-type":[{"value":"2073-4409","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,11,3]]}}}