Effects of storage time and temperature on coagulation factor and natural anticoagulant activities

Effects of storage time and temperature on coagulation factor and natural anticoagulant activities | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Effects of storage time and temperature on coagulation factor and natural anticoagulant activities Ryosuke Nakanishi, Tasuku Sakayori, Daichi Matsui, Mai Kono, Minase Maki, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5314705/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Mar, 2025 Read the published version in Scientific Reports → Version 1 posted 12 You are reading this latest preprint version Abstract Coagulation test results are influenced by many pre-analytical variables, including clinical sample storage conditions. This study investigated the effects of storage time and temperature on coagulation factor (FII, FV, FVII, FX, FVIII, FIX, FXI, FXII, and FXIII) and natural anticoagulant (antithrombin, protein C, and free protein S) activities. Blood samples from healthy individuals were centrifuged and plasma was tested after room temperature (18 to 25°C) or refrigerated (2 to 8°C) for 2, 3, 4, and 5 hours, or after frozen storage (-15 to -25°C) for 1, 2, 3, and 4 months. For all storage conditions, the mean percentage change from baseline in evaluated activities was 15%. For FV and FVIII activities, the mean percentage change was < 15% after frozen storage at -75 to -85°C for 1, 2, 3, and 4 months. These labile factors are associated with rapid decreases in plasma levels in vitro . To ensure appropriate clinical testing results, especially for FV and FVIII activity, storage condition and time should be critically controlled. Biological sciences/Immunology/Coagulation system Biological sciences/Biotechnology/Assay systems Biological sciences/Biological techniques/Analytical biochemistry/Biochemical assays Biological sciences/Drug discovery/Diagnostics Health sciences/Health care/Diagnosis/Laboratory techniques and procedures Physical sciences/Chemistry/Analytical chemistry/Medical and clinical diagnostics pre-analytical variable coagulation tests stability storage conditions Clinical and Laboratory Standards Institute Figures Figure 1 Figure 2 Introduction Numerous pre-analytical variables may influence coagulation test results. The most important pre-analytical variables include sample storage time and temperature [ 1 , 2 ]. Erroneous results, influenced by pre-analytical variables, can significantly impact diagnosis and treatment, and consequently patient outcomes [ 3 ]. The Clinical and Laboratory Standards Institute (CLSI) H21-ED6 guideline recommends acceptable storage times for plasma samples at various storage conditions (room temperature, refrigerated, or frozen) (Table 1 ) [ 4 ]. The main text of the CLSI H21-ED6 guideline states that samples for prothrombin time (PT), fibrinogen, and D-dimer tests can be stored at room temperature for up to 24 hours after blood collection, and samples for activated partial thromboplastin time (APTT) (not for heparin monitoring) can be stored at room temperature for up to 8 hours. For other coagulation factor and natural anticoagulant activities, the CLSI H21-ED6 guideline suggests that additional studies are needed to determine whether extending the acceptable storage time beyond the existing default of 4 hours is safe. However, evidence provided in the CLSI H21-ED6 guideline is limited. Appendix D of the CLSI H21-ED6 guideline lists the acceptable storage times for each parameter (coagulation factors and natural anticoagulants) at room temperature and when refrigerated, but this can be considered preliminary evidence. Table 1 Comparison of acceptable storage times for FII, FV, FVII, FX, FVIII, FIX, FXI, FXII, FXIII, AT, PC, and free PS activities between the CLSI guideline and this study Acceptable storage time (main text of the CLSI H21-ED6 guideline) Acceptable storage time (this study) Factor Room temperature (15 to 25°C) Refrigerated (2 to 8°C) Frozen (≤ -20°C) Frozen (≤ -70°C) Room temperature (18 to 25°C) Refrigerated (2 to 8°C) Frozen (-15 to -25°C) Frozen (-75 to -85°C) FII (%) 4 hours 4 hours 3 months 18 months 5 hours 5 hours 4 months - FV (%) 4 hours 4 hours 3 months 18 months 5 hours 5 hours unacceptable 4 months FVII (%) 4 hours 4 hours 3 months 18 months 5 hours 5 hours 4 months - FX (%) 4 hours 4 hours 3 months 18 months 5 hours 5 hours 4 months - FVIII (%) 4 hours 4 hours 3 months 18 months 3 hours 4 hours 1 month 4 months FIX (%) 4 hours 4 hours 3 months 18 months 5 hours 5 hours 4 months - FXI (%) 4 hours 4 hours 3 months 18 months 5 hours 5 hours 4 months - FXII (%) 4 hours 4 hours 3 months 18 months 5 hours 5 hours 4 months - FXIII (%) 4 hours 4 hours 3 months 18 months 5 hours 5 hours 4 months - AT (%) 4 hours 4 hours 3 months 18 months 5 hours 5 hours 4 months - PC (%) 4 hours 4 hours 3 months 18 months 5 hours 5 hours 4 months - Free PS (%) 4 hours 4 hours 3 months 18 months 5 hours 5 hours 4 months - Abbreviations: FII, factor II; FV, factor V; FVII, factor VII; FX, factor X; FVIII, factor VIII; FIX, factor IX; FXI, factor XI; FXII, factor XII; FXIII, factor XIII; AT, antithrombin; PC, protein C; Free PS, free protein S. Few studies have suggested acceptable storage times at room temperature and under refrigeration for coagulation factor activities [ 5 – 8 ]. Previous studies have indicated that the acceptable storage times at room temperature and under refrigeration for FV and FVIII activities are shorter than those for other coagulation factors [ 5 – 8 ]. When samples are frozen, acceptable storage times for PT, APTT, fibrinogen antigen, and Clauss fibrinogen have been considered in the main text of the CLSI H21-ED6 guideline. The CLSI H21-ED6 guideline recommends that plasma samples, when used for coagulation testing, be stored frozen for up to 3 months at ≤ -20°C and up to 18 months at ≤ -70°C. However, the evidence supporting these recommendations is limited. The acceptable storage times for each parameter (coagulation factors and natural anticoagulants) are detailed in Appendix D of the CLSI H21-ED6 guideline. However, the CLSI H21-ED6 guideline states that these acceptable storage times may exceed the general recommendations for frozen storage. Previous studies have reported that acceptable frozen storage times for FVIII and FIX activities are shorter than those for other coagulation tests [ 9 ]. However, the findings of the few studies on frozen storage of samples must be validated. To ensure accurate measurement data using a combination of reagents and equipment, sample stability must be considered. This study investigated the effects of storage time and temperature, including room temperature (18 to 25°C), refrigerated (2 to 8°C), and frozen (-15 to -25°C) storage, in the clinical laboratory environment on coagulation factor (FII, FV, FVII, FX, FVIII, FIX, FXI, FXII, and FXIII) and natural anticoagulant (antithrombin [AT], protein [P] C, and free PS) activities. The study also focused on coagulation proteins not previously studied in all storage conditions, such as FXIII (room temperature, refrigerated, and frozen), AT (room temperature and refrigerated), PC (room temperature and refrigerated), and free PS (room temperature, refrigerated, and frozen). Results Figure 1 presents the trend plot for the mean percentage changes in FII, FV, FVII, FX, FVIII, FIX, FXI, FXII, FXIII, AT, PC, and free PS activities after room temperature (Fig. 1 a) or refrigerated (Fig. 1 b) storage for 2, 3, 4, and 5 hours, or after frozen (-15 to -25°C) (Fig. 1 c) storage for 1, 2, 3, and 4 months, compared with their respective baseline values (0 hours and 0 months). The values between the analytes at each storage temperature are shown in Supplementary Table 1. After room temperature (Fig. 1 a) or refrigerated (Fig. 1 b) storage, the mean percentage changes in all factor and natural anticoagulant activities, except FVIII, were less than 15% within the time frame of this study. FV activity after storage for 5 hours at room temperature decreased from 107–95%, and the mean percentage change was − 11%. The stability of FVIII activity after room temperature and refrigerated storage was lower than that of the activities of other factors. The mean percentage changes in FVIII activity after room-temperature storage for more than 2 hours or after refrigerated storage for more than 4 hours exceeded 15%, with a decrease in FVIII activity from 110–92% when stored for 2 hours at room temperature and from 110–89% when refrigerated for 4 hours. The mean percentage changes were − 16% and − 19%, respectively. FVIII activity after room temperature or refrigerated storage for 5 hours decreased from 110–63% and 85%, respectively. The mean percentage changes were − 43% and − 23%, respectively. Since the mean percentage change in FVIII activity after room temperature or refrigerated storage exceeded 15%, additional measurements were conducted every 0.5 hours between 1 and 4.5 hours under the 2 conditions of storage to investigate the detailed stability of FVIII activity. To obtain more accurate data, the time from blood collection to measurement was shortened. Figure 2 a shows the trend plot for the mean percentage changes in FVIII activity after room-temperature or refrigerated storage for 1, 1.5, 2, 2.5, 3, 3.5, 4, and 4.5 hours. The mean percentage change in FVIII activity after room-temperature storage for 3 hours or after refrigerated storage for 4 hours was less than 15%. FVIII activity after room-temperature storage for 3.5 hours decreased from 137–115%, and the mean percentage change was − 16%. FVIII activity after refrigerated storage for 4.5 hours decreased from 137–114%, and the mean percentage change was − 17%. The values in FVIII activity compared with baseline measurements (0 hours) are shown in Supplementary Table 2. After frozen storage (-15 to -25°C) (Fig. 1 c), the mean percentage changes for all coagulation factor activities and natural anticoagulant activities, except FV and FVIII activities, were less than 15% within the time frame of this study. The stability of FV and FVIII activities after frozen storage (-15 to -25°C) was lower than that of other factor activities. The mean percentage change in FV activity after frozen (-15 to -25°C) storage for more than 1 month and FVIII activity after frozen (-15 to -25°C) storage for more than 2 months exceeded 15%. FV activity after 1 month of frozen storage (-15 to -25°C) decreased from 79–66%, with a mean percentage change of -16%. FVIII activity after 2 months of frozen storage (-15 to -25°C) decreased from 151–123%, with a mean percentage change of -19%. Therefore, to investigate the detailed stability of FV and FVIII activity after frozen storage, additional measurements were conducted on the samples after frozen storage (-75 to -85°C). Figure 2 b shows the trend plot for the mean percentage changes in FV and FVIII activities after frozen storage at ≤-75°C for 1, 2, 3, and 4 months. The mean percentage changes in FV and FVIII activities measured after frozen storage at ≤-75°C for 1, 2, 3, and 4 months were less than 15% for all times. FV and FVIII activities after frozen storage (-75 to -85°C) for 4 months decreased from 113–104% and from 137–127%, respectively. The samples for FV and FVIII can be safely stored in this time frame when plasma is stored frozen at ≤-75°C. All results in FV and FVIII activities after frozen storage (-75 to -85°C) for 1, 2, 3, and 4 months are shown in Supplementary Table 2. Discussion Table 1 presents a summary of the acceptable storage times according to the main text of the CLSI H21-ED6 guideline and our findings. First, we focused on room-temperature and refrigerated storage. As shown in Table 1 , the acceptable storage time at room temperature for FVIII activity, an important laboratory test parameter for the diagnosis of hemophilia and related disorders, was shorter than that recommended in the main text of the CLSI H21-ED6 guideline. The acceptable room-temperature and refrigerated storage times for FVIII activity were shorter than those for other factors, as previously reported in studies using reagents and equipment from other manufacturers [ 5 , 7 , 8 ]. The storage times for FVIII activity in previous studies were shorter than the acceptable storage time described in the main text of the CLSI H21-ED6 guideline, supporting the results of the present study. However, this study confirmed that the acceptable room-temperature and refrigerated storage times for FV activity are the same as the acceptable storage times for other coagulation factor and natural anticoagulant activities, excluding FVIII activity. However, FV activity after storage at room temperature for 5 hours decreased the most among coagulation factor and natural anticoagulant activities, excluding FVIII activity. FV activity at room temperature tended to decrease. FV activity has long been thought to be unstable with respect to storage time and temperature in clinical laboratory tests. A recent study using other manufacturers’ reagents and equipment demonstrated that the acceptable storage time for FV activity at room temperature is shorter than that of other coagulation factors (FII, FVII, FX, FIX, FXI, and FXII) [ 8 ]. The mean percentage change in FVIII activity after storage for 5 hours at room temperature in this study was − 43%, indicating that the activity decreased by almost half. Further studies are necessary for samples with originally low activity, such as hemophilia samples. These results suggest that samples from patients with hemophilia (especially for the diagnostic testing of mild hemophilia) pose a risk of misdiagnosis, which could impact patient management. Next, this study found that storing plasma frozen at -15 to -25°C is not suitable for the stability of FV and FVIII activities. Previous studies using other manufacturers’ reagents have suggested that FVIII and FIX activities decreased when plasma is stored frozen at -20°C [ 9 ]. FIX activity was stable within the timeframe of this study, whereas FV and FVIII activities were more unstable than those of the other coagulation factors and natural anticoagulants. Although further studies, such as those including larger sample sizes, using patient samples, or using other manufacturers’ reagents, are necessary, based on the results of previous studies and this study, caution must be exercised when storing samples for FV, FVIII, and FIX activity testing at freezing temperatures. For FV and FVIII activities, which were not stable when plasma was frozen at -15 to -25°C, we changed the storage temperature from freezing at -15 to -25°C to freezing at -75 to -85°C, and we obtained additional data. From the results shown in Fig. 2 b, FV and FVIII activities after frozen storage at -75 to -85°C were stable within the time frame of this study. While the CLSI H21-ED6 guideline does not mention this, the present study recommends storing samples in a deep freezer for measuring FV and FVIII activities when the sample is stored frozen. In terms of long-term storage, our results differ from those of a previous study, which reported that FV and FVIII activities were stable for 6 and 3 months, respectively, after frozen storage at -24 ± 2°C [ 10 ]. However, the previous study involved samples collected via plasmapheresis, which differs from the sample collection method used in the present study. The blood collection method used in this study (brachial vein blood collection) was similar to that commonly used in routine clinical laboratory tests. The differences in blood collection methods are assumed to have influenced the results. Although further studies on the stability of FV and FVIII activities are required, long-term storage is not recommended. To obtain appropriate data, measurements must be performed within a short storage period after blood collection. Moreover, new insights into the effects of storage time and temperature on coagulation factors and natural anticoagulants were obtained for FXIII (room temperature, refrigerated, and frozen), AT (room temperature and refrigerated), PC (room temperature and refrigerated), and free PS (room temperature, refrigerated, and frozen), which have not been previously reported. The coagulation factors and natural anticoagulants remained stable within these timeframes under all storage conditions. This study found that the acceptable storage time and temperature varied for each coagulation factor and natural anticoagulant. To provide appropriate measurement data in clinical practice, storage times and temperatures should be critically controlled. In summary, this study showed that samples for the measurement of FII, FV, FVII, FX, FIX, FXI, FXII, FXIII, AT, PC, and free PS activities can be stored for up to 5 hours at room temperature or under refrigeration, whereas samples for the measurement of FVIII activity can be stored for only 3 and 4 hours, respectively, at the same temperatures. Also, our findings indicate that samples for the measurement of FII, FVII, FX, FIX, FXI, FXII, FXIII, AT, PC, and free PS activities could be stored frozen at -15 to -25°C for up to 4 months. FV and FVIII activities required frozen storage at -75 to -85°C for the same duration. Nonetheless, this study used plasma samples obtained from only a limited number of healthy individuals. Furthermore, the stability of coagulation factors and natural anticoagulant activities may be affected by patients' clinical states or medications. Therefore, the conclusions of this study should be substantiated by further studies involving different study populations. Methods Ethics statement This study was approved by the Sysmex Corporation Ethics Committee (approval No., 2021-24). All methods were performed in accordance with Ethical Guidelines for Medical and Biological Research Involving Human Subjects. The internal volunteers provided written informed consent for the use of their samples in this study. Participating centers This study was conducted at Sysmex Corporation (Kobe, Japan). Samples were measured using AUTOMATED BLOOD COAGULATION ANALYZER CS-5100 (Sysmex Corp., Kobe, Japan) and Automated Blood Coagulation Analyzer CN-6000 (Sysmex Corp., Kobe, Japan) with Sysmex (Sysmex Corp., Kobe, Japan) and HYPHEN reagents (HYPHEN BioMed, Paris, France). Plasma samples Healthy individuals (n = 78) were recruited from internal volunteers at the Sysmex Corporation for coagulation factors (FII, FV, FVII, FX, FVIII, FIX, FXI, FXII, and FXIII) and natural anticoagulants (AT, PC, and free PS). Plasma samples were prepared according to the consensus established by Ieko et al. [ 11 ]. From each volunteer, 9 mL (4.5 mL x 2) blood samples were collected in Venoject II® tubes (Terumo Corp, Tokyo, Japan) containing one-tenth volume of 0.105 M buffered sodium citrate (approximately 3.2%). Whole blood was centrifuged at 1500 × g for 15 minutes at room temperature. The plasma obtained was collected using plastic pipettes and transferred to polystyrene tubes, which were then capped. For each coagulation factor and natural anticoagulant, one group of samples (n = 3) was divided between room temperature (18 to 25°C) and refrigerated storage (2 to 8°C), while the other group (n = 3) was frozen (-15 to -25°C). A portion of each sample was analyzed, and the results served as the baseline (0 hours or 0 months). The other samples were tested under the following conditions from baseline measurement: after storage for 2, 3, 4, and 5 hours at room temperature and under refrigeration and for 1, 2, 3, and 4 months at frozen (-15 to -25°C). The refrigerators and freezers used to store the samples were monitored. The samples stored frozen at -15 to -25°C were thawed in a water bath for 15 minutes at 37°C before use. Assays The plasma samples were measured using the chromogenic method for FVIII, FXIII, AT, PC, and free PS and the clotting method for FII, FV, FVII, FX, FIX, FXI, and FXII. Measurements were conducted using CS-5100 for AT and CN-6000 for FII, FV, FVII, FX, FVIII, FIX, FXI, FXII, FXIII, PC, and free PS. HYPHEN BioMed reagents included Prothrombin Deficient Plasma, Factor V Deficient Plasma, Factor VII Deficient Plasma, Factor X Deficient Plasma, PT-Phen™ LRT, Revohem™ FVIII Chromogenic, Factor IX Deficient Plasma, Factor XI Deficient Plasma, Factor XII Deficient Plasma, BIOPHEN™ Factor XIII, Revohem™ AT, Revohem™ Protein C, and LIAPHEN™ Free Protein S. Sysmex reagents included Revohem™ APTT SLA and Revohem™ 0.025 M Calcium Chloride Solution. The results are expressed as percentages. Statistical analyses The measured coagulation factor and natural anticoagulant activities are reported as mean ± standard deviation (SD). To assess the stability of these activities, the percentage changes from the baseline values were calculated ([result at storage time X - result at baseline] / result at baseline) and averaged at each time point. A mean percentage change of ≤ 15% compared to the baseline values was considered indicative of acceptable storage. When the mean percentage change in the coagulation factor activity tested after storage at room temperature or under refrigeration exceeded 15% compared with the baseline values, additional samples (n = 3) were collected for the coagulation factor activity test and measured after every 30 minutes for 1–4.5 hours at the same storage temperature. When the mean percentage change in coagulation factor activity after frozen storage (-15 to -25°C) exceeded 15% compared with the baseline values, additional samples (n = 3) were collected for the coagulation factor activity test and measured after storage for 1, 2, 3, and 4 months at frozen (-75 to -85°C). When the mean percentage change in the additionally measured results exceeded 15% compared with the baseline values, the measurement was considered complete. The freezers were monitored while storing the samples. The samples stored frozen at -75 to -85°C were thawed in a water bath for 15 minutes at 37°C before use. Declarations Competing Interests R.N., T.S., D.M., M.K., M.M. and H.M. are employees of Sysmex Corporation. C.D. is an employee of HYPHEN BioMed. Y.K. is an outside adviser for Sysmex Corporation. Author Contribution R.N. designed and performed the experiments, analyzed the data, and drafted the manuscript. D.M., M.K., M.M., and C.D. designed the experiments. Y.K. interpreted the data and revised the manuscript. T.S. and H.M. supervised the study and revised the manuscript. All the authors critically reviewed and approved the final version of the manuscript. Acknowledgement This study was supported by the Sysmex Corporation. Data Availability The datasets analyzed during this study are available from the corresponding author on reasonable request. References Gosselin, R. C. & Marlar, R. A. Preanalytical variables in coagulation testing: setting the stage for accurate results. Semin. Thromb. Hemost. 45 , 433-448 (2019). Adcock, D. M., Favaloro, E. J. & Lippi, G. Critical pre-examination variables in the hemostasis laboratory and their quality indicators. Clin. Biochem. 49 , 1315–1320 (2016). Favaloro, E. J., Lippi, G. & Adcock, D. M. Preanalytical and postanalytical variables: the leading causes of diagnostic error in hemostasis? Semin. Thromb. Hemost. 34 , 612-634 (2008). CLSI. Collection, Transport, and Processing of Blood Specimens for Testing Plasma-Based Coagulation Assays- 6th ed. CLSI document H21-ED6 Wayne (Clinical and Lab. Standards Inst., PA, 2024). Feng, L., Zhao, Y., Zhao, H. & Shao, Z. Effects of storage time and temperature on coagulation tests and factors in fresh plasma. Sci. Rep. 4 , 3868 (2014). Zhao, Y., Feng, G. & Feng, L. Effects of pre-analytical storage time, temperature, and freeze-thaw times on coagulation factors activities in citrate-anticoagulated plasma. Ann. Transl. Med. 6 , 456 (2018). Böhm, M. et al . Cold storage of citrated whole blood induces drastic time-dependent losses in factor VIII and von Willebrand factor: potential for misdiagnosis of haemophilia and von Willebrand disease. Blood Coagul. Fibrinolysis 17 , 39-45 (2006). Linskens, E. A. & Devreese, K. M. J. Pre-analytical stability of coagulation parameters in plasma stored at room temperature. Int. J. Lab. Hematol. 40 , 292-303 (2018). Zhao, Y. et al . Effects of preanalytical frozen storage time and temperature on screening coagulation tests and factors VIII and IX activity. Sci. Rep. 7 , 12179 (2017). Woodhams, B., Girardot, O., Blanco, M. J., Colesse, G. & Gourmelin, Y. Stability of coagulation proteins in frozen plasma. Blood Coagul. Fibrinolysis 12 , 229-236 (2001). Ieko, M. et al . Expert consensus regarding standardization of sample preparation for clotting time assays. Int. J. Hematol. 112, 614-620 (2020). Additional Declarations Competing interest reported. R.N., T.S., D.M., M.K., M.M. and H.M. are employees of Sysmex Corporation. C.D. is an employee of HYPHEN BioMed. Y.K. is an outside adviser for Sysmex Corporation. Supplementary Files SupplementaryTable.xlsx Cite Share Download PDF Status: Published Journal Publication published 28 Mar, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 05 Dec, 2024 Reviews received at journal 04 Dec, 2024 Reviews received at journal 29 Nov, 2024 Reviews received at journal 27 Nov, 2024 Reviewers agreed at journal 19 Nov, 2024 Reviewers agreed at journal 18 Nov, 2024 Reviewers agreed at journal 18 Nov, 2024 Reviewers invited by journal 18 Nov, 2024 Editor assigned by journal 18 Nov, 2024 Editor invited by journal 07 Nov, 2024 Submission checks completed at journal 07 Nov, 2024 First submitted to journal 22 Oct, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5314705","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":380033709,"identity":"7c0df79a-790d-4aae-8c2f-ce971185f638","order_by":0,"name":"Ryosuke Nakanishi","email":"","orcid":"","institution":"Sysmex (Japan)","correspondingAuthor":false,"prefix":"","firstName":"Ryosuke","middleName":"","lastName":"Nakanishi","suffix":""},{"id":380033710,"identity":"02330c20-6bbc-439c-b236-b6d39cc1593d","order_by":1,"name":"Tasuku Sakayori","email":"data:image/png;base64,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","orcid":"","institution":"Sysmex (Japan)","correspondingAuthor":true,"prefix":"","firstName":"Tasuku","middleName":"","lastName":"Sakayori","suffix":""},{"id":380033711,"identity":"16bf98bf-7b8c-4724-8a44-f314e227182d","order_by":2,"name":"Daichi Matsui","email":"","orcid":"","institution":"Sysmex (Japan)","correspondingAuthor":false,"prefix":"","firstName":"Daichi","middleName":"","lastName":"Matsui","suffix":""},{"id":380033712,"identity":"47377f40-9ffd-4796-bfc6-ccd68b788623","order_by":3,"name":"Mai Kono","email":"","orcid":"","institution":"Sysmex (Japan)","correspondingAuthor":false,"prefix":"","firstName":"Mai","middleName":"","lastName":"Kono","suffix":""},{"id":380033713,"identity":"07be57ce-e1ba-4a85-868c-0d796b408552","order_by":4,"name":"Minase Maki","email":"","orcid":"","institution":"Sysmex (Japan)","correspondingAuthor":false,"prefix":"","firstName":"Minase","middleName":"","lastName":"Maki","suffix":""},{"id":380033714,"identity":"a8fe60f5-2388-4146-ab71-662d6414d884","order_by":5,"name":"Claire Dunois","email":"","orcid":"","institution":"HYPHEN BioMed","correspondingAuthor":false,"prefix":"","firstName":"Claire","middleName":"","lastName":"Dunois","suffix":""},{"id":380033715,"identity":"91bd85f7-5b8e-4608-be07-6bf7c4b905f5","order_by":6,"name":"Yutaka Komiyama","email":"","orcid":"","institution":"Hokuriku University","correspondingAuthor":false,"prefix":"","firstName":"Yutaka","middleName":"","lastName":"Komiyama","suffix":""},{"id":380033716,"identity":"08327e5d-dd48-48bb-9665-646528b69c31","order_by":7,"name":"Hiromi Morisaki","email":"","orcid":"","institution":"Sysmex (Japan)","correspondingAuthor":false,"prefix":"","firstName":"Hiromi","middleName":"","lastName":"Morisaki","suffix":""}],"badges":[],"createdAt":"2024-10-23 00:53:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5314705/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5314705/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-95389-w","type":"published","date":"2025-03-28T15:57:16+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":69820384,"identity":"db5e6ad5-bbf6-4768-9164-3e1b047c19ae","added_by":"auto","created_at":"2024-11-25 14:10:04","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":123995,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTrend plot for mean percentage changes in coagulation and anticoagulant factor activities in sample after storage\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTrend plot for mean percentage changes in FII, FV, FVII, FX, FVIII, FIX, FXI, FXII, FXIII, AT, PC, and free PS activities in samples after storage for 2, 3, 4, and 5 hours at room temperature (18 to 25 °C) (\u003cstrong\u003ea\u003c/strong\u003e); 2, 3, 4, and 5 hours under refrigeration (2 to 8 °C) (\u003cstrong\u003eb\u003c/strong\u003e); and 1, 2, 3, and 4 months at frozen (-15 to -25 °C) (\u003cstrong\u003ec\u003c/strong\u003e), compared with the baseline results. The two dotted lines in the figures represent ± 15%.\u003c/p\u003e\n\u003cp\u003eAbbreviations: FII, factor II; FV, factor V; FVII, factor VII; FX, factor X; FVIII, factor VIII; FIX, factor IX; FXI, factor XI; FXII, factor XII; FXIII, factor XIII; AT, antithrombin; PC, protein C; Free PS, free protein S.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5314705/v1/9cb1e58352f664fadbf8c797.jpg"},{"id":69820902,"identity":"1f657f71-53c8-4f81-8f87-d2aabd488924","added_by":"auto","created_at":"2024-11-25 14:18:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":55959,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTrend plot for mean percentage changes in FV and FVIII activities in additional samples after storage\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Trend plot for mean percentage changes in FVIII activities in additional samples after storage for 1, 1.5, 2, 2.5, 3, 3.5, 4, and 4.5 hours at room temperature (18 to 25 °C) and under refrigeration (2 to 8 °C), compared with the baseline results. (\u003cstrong\u003eb\u003c/strong\u003e) Trend plot for mean percentage changes in FV and FVIII activities in samples after frozen (-75 to -85 °C) storage for 1, 2, 3, and 4 months, compared with the baseline results. The two dotted lines in the figures represent ± 15%.\u003c/p\u003e\n\u003cp\u003eAbbreviations: FV, factor V; FVIII, factor VIII.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5314705/v1/742106326ec5793dcd0cbb56.png"},{"id":79604981,"identity":"c4a09ccb-cf13-4d4e-b8d9-894b2965145d","added_by":"auto","created_at":"2025-03-31 16:09:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":797212,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5314705/v1/1fb398c7-4077-4117-bade-d557650e8dfe.pdf"},{"id":69820381,"identity":"9679f3cd-c836-4690-87a6-d47e5945a355","added_by":"auto","created_at":"2024-11-25 14:10:02","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":20138,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5314705/v1/4ade3acaf7964b2272c88bcd.xlsx"}],"financialInterests":"Competing interest reported. R.N., T.S., D.M., M.K., M.M. and H.M. are employees of Sysmex Corporation. C.D. is an employee of HYPHEN BioMed. Y.K. is an outside adviser for Sysmex Corporation.","formattedTitle":"Effects of storage time and temperature on coagulation factor and natural anticoagulant activities","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNumerous pre-analytical variables may influence coagulation test results. The most important pre-analytical variables include sample storage time and temperature [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Erroneous results, influenced by pre-analytical variables, can significantly impact diagnosis and treatment, and consequently patient outcomes [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe Clinical and Laboratory Standards Institute (CLSI) H21-ED6 guideline recommends acceptable storage times for plasma samples at various storage conditions (room temperature, refrigerated, or frozen) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The main text of the CLSI H21-ED6 guideline states that samples for prothrombin time (PT), fibrinogen, and D-dimer tests can be stored at room temperature for up to 24 hours after blood collection, and samples for activated partial thromboplastin time (APTT) (not for heparin monitoring) can be stored at room temperature for up to 8 hours. For other coagulation factor and natural anticoagulant activities, the CLSI H21-ED6 guideline suggests that additional studies are needed to determine whether extending the acceptable storage time beyond the existing default of 4 hours is safe. However, evidence provided in the CLSI H21-ED6 guideline is limited. Appendix D of the CLSI H21-ED6 guideline lists the acceptable storage times for each parameter (coagulation factors and natural anticoagulants) at room temperature and when refrigerated, but this can be considered preliminary evidence.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of acceptable storage times for FII, FV, FVII, FX, FVIII, FIX, FXI, FXII, FXIII, AT, PC, and free PS activities between the CLSI guideline and this study\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e \u003cp\u003eAcceptable storage time \u003c/p\u003e \u003cp\u003e(main text of the CLSI H21-ED6 guideline)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c9\" namest=\"c6\"\u003e \u003cp\u003eAcceptable storage time (this study)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFactor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRoom temperature \u003c/p\u003e \u003cp\u003e(15 to 25\u0026deg;C)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRefrigerated\u003c/p\u003e \u003cp\u003e(2 to 8\u0026deg;C)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFrozen\u003c/p\u003e \u003cp\u003e(\u0026le; -20\u0026deg;C)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFrozen\u003c/p\u003e \u003cp\u003e(\u0026le; -70\u0026deg;C)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRoom temperature \u003c/p\u003e \u003cp\u003e(18 to 25\u0026deg;C)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eRefrigerated\u003c/p\u003e \u003cp\u003e(2 to 8\u0026deg;C)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eFrozen \u003c/p\u003e \u003cp\u003e(-15 to -25\u0026deg;C)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eFrozen \u003c/p\u003e \u003cp\u003e(-75 to -85\u0026deg;C)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFII (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4 hours\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4 hours\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5 hours\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5 hours\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFV (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4 hours\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4 hours\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5 hours\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5 hours\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eunacceptable\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e4 months\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFVII (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4 hours\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4 hours\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5 hours\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5 hours\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFX (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4 hours\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4 hours\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5 hours\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5 hours\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFVIII (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4 hours\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4 hours\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3 hours\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4 hours\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1 month\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e4 months\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFIX (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4 hours\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4 hours\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5 hours\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5 hours\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFXI (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4 hours\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4 hours\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5 hours\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5 hours\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFXII (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4 hours\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4 hours\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5 hours\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5 hours\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFXIII (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4 hours\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4 hours\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5 hours\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5 hours\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAT (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4 hours\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4 hours\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5 hours\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5 hours\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePC (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4 hours\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4 hours\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5 hours\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5 hours\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFree PS (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4 hours\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4 hours\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5 hours\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5 hours\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"9\"\u003eAbbreviations: FII, factor II; FV, factor V; FVII, factor VII; FX, factor X; FVIII, factor VIII; FIX, factor IX; FXI, factor XI; FXII, factor XII; FXIII, factor XIII; AT, antithrombin; PC, protein C; Free PS, free protein S.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eFew studies have suggested acceptable storage times at room temperature and under refrigeration for coagulation factor activities [\u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Previous studies have indicated that the acceptable storage times at room temperature and under refrigeration for FV and FVIII activities are shorter than those for other coagulation factors [\u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e When samples are frozen, acceptable storage times for PT, APTT, fibrinogen antigen, and Clauss fibrinogen have been considered in the main text of the CLSI H21-ED6 guideline. The CLSI H21-ED6 guideline recommends that plasma samples, when used for coagulation testing, be stored frozen for up to 3 months at \u0026le; -20\u0026deg;C and up to 18 months at \u0026le; -70\u0026deg;C. However, the evidence supporting these recommendations is limited. The acceptable storage times for each parameter (coagulation factors and natural anticoagulants) are detailed in Appendix D of the CLSI H21-ED6 guideline. However, the CLSI H21-ED6 guideline states that these acceptable storage times may exceed the general recommendations for frozen storage.\u003c/p\u003e \u003cp\u003ePrevious studies have reported that acceptable frozen storage times for FVIII and FIX activities are shorter than those for other coagulation tests [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. However, the findings of the few studies on frozen storage of samples must be validated.\u003c/p\u003e \u003cp\u003eTo ensure accurate measurement data using a combination of reagents and equipment, sample stability must be considered. This study investigated the effects of storage time and temperature, including room temperature (18 to 25\u0026deg;C), refrigerated (2 to 8\u0026deg;C), and frozen (-15 to -25\u0026deg;C) storage, in the clinical laboratory environment on coagulation factor (FII, FV, FVII, FX, FVIII, FIX, FXI, FXII, and FXIII) and natural anticoagulant (antithrombin [AT], protein [P] C, and free PS) activities. The study also focused on coagulation proteins not previously studied in all storage conditions, such as FXIII (room temperature, refrigerated, and frozen), AT (room temperature and refrigerated), PC (room temperature and refrigerated), and free PS (room temperature, refrigerated, and frozen).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e presents the trend plot for the mean percentage changes in FII, FV, FVII, FX, FVIII, FIX, FXI, FXII, FXIII, AT, PC, and free PS activities after room temperature (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea) or refrigerated (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb) storage for 2, 3, 4, and 5 hours, or after frozen (-15 to -25\u0026deg;C) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec) storage for 1, 2, 3, and 4 months, compared with their respective baseline values (0 hours and 0 months). The values between the analytes at each storage temperature are shown in Supplementary Table\u0026nbsp;1.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAfter room temperature (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea) or refrigerated (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb) storage, the mean percentage changes in all factor and natural anticoagulant activities, except FVIII, were less than 15% within the time frame of this study. FV activity after storage for 5 hours at room temperature decreased from 107\u0026ndash;95%, and the mean percentage change was \u0026minus;\u0026thinsp;11%.\u003c/p\u003e \u003cp\u003eThe stability of FVIII activity after room temperature and refrigerated storage was lower than that of the activities of other factors. The mean percentage changes in FVIII activity after room-temperature storage for more than 2 hours or after refrigerated storage for more than 4 hours exceeded 15%, with a decrease in FVIII activity from 110\u0026ndash;92% when stored for 2 hours at room temperature and from 110\u0026ndash;89% when refrigerated for 4 hours. The mean percentage changes were \u0026minus;\u0026thinsp;16% and \u0026minus;\u0026thinsp;19%, respectively. FVIII activity after room temperature or refrigerated storage for 5 hours decreased from 110\u0026ndash;63% and 85%, respectively. The mean percentage changes were \u0026minus;\u0026thinsp;43% and \u0026minus;\u0026thinsp;23%, respectively.\u003c/p\u003e \u003cp\u003eSince the mean percentage change in FVIII activity after room temperature or refrigerated storage exceeded 15%, additional measurements were conducted every 0.5 hours between 1 and 4.5 hours under the 2 conditions of storage to investigate the detailed stability of FVIII activity. To obtain more accurate data, the time from blood collection to measurement was shortened.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea shows the trend plot for the mean percentage changes in FVIII activity after room-temperature or refrigerated storage for 1, 1.5, 2, 2.5, 3, 3.5, 4, and 4.5 hours. The mean percentage change in FVIII activity after room-temperature storage for 3 hours or after refrigerated storage for 4 hours was less than 15%. FVIII activity after room-temperature storage for 3.5 hours decreased from 137\u0026ndash;115%, and the mean percentage change was \u0026minus;\u0026thinsp;16%. FVIII activity after refrigerated storage for 4.5 hours decreased from 137\u0026ndash;114%, and the mean percentage change was \u0026minus;\u0026thinsp;17%. The values in FVIII activity compared with baseline measurements (0 hours) are shown in Supplementary Table\u0026nbsp;2.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAfter frozen storage (-15 to -25\u0026deg;C) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec), the mean percentage changes for all coagulation factor activities and natural anticoagulant activities, except FV and FVIII activities, were less than 15% within the time frame of this study. The stability of FV and FVIII activities after frozen storage (-15 to -25\u0026deg;C) was lower than that of other factor activities. The mean percentage change in FV activity after frozen (-15 to -25\u0026deg;C) storage for more than 1 month and FVIII activity after frozen (-15 to -25\u0026deg;C) storage for more than 2 months exceeded 15%. FV activity after 1 month of frozen storage (-15 to -25\u0026deg;C) decreased from 79\u0026ndash;66%, with a mean percentage change of -16%. FVIII activity after 2 months of frozen storage (-15 to -25\u0026deg;C) decreased from 151\u0026ndash;123%, with a mean percentage change of -19%. Therefore, to investigate the detailed stability of FV and FVIII activity after frozen storage, additional measurements were conducted on the samples after frozen storage (-75 to -85\u0026deg;C). Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb shows the trend plot for the mean percentage changes in FV and FVIII activities after frozen storage at \u0026le;-75\u0026deg;C for 1, 2, 3, and 4 months. The mean percentage changes in FV and FVIII activities measured after frozen storage at \u0026le;-75\u0026deg;C for 1, 2, 3, and 4 months were less than 15% for all times. FV and FVIII activities after frozen storage (-75 to -85\u0026deg;C) for 4 months decreased from 113\u0026ndash;104% and from 137\u0026ndash;127%, respectively. The samples for FV and FVIII can be safely stored in this time frame when plasma is stored frozen at \u0026le;-75\u0026deg;C. All results in FV and FVIII activities after frozen storage (-75 to -85\u0026deg;C) for 1, 2, 3, and 4 months are shown in Supplementary Table\u0026nbsp;2.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e presents a summary of the acceptable storage times according to the main text of the CLSI H21-ED6 guideline and our findings.\u003c/p\u003e \u003cp\u003eFirst, we focused on room-temperature and refrigerated storage. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the acceptable storage time at room temperature for FVIII activity, an important laboratory test parameter for the diagnosis of hemophilia and related disorders, was shorter than that recommended in the main text of the CLSI H21-ED6 guideline. The acceptable room-temperature and refrigerated storage times for FVIII activity were shorter than those for other factors, as previously reported in studies using reagents and equipment from other manufacturers [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The storage times for FVIII activity in previous studies were shorter than the acceptable storage time described in the main text of the CLSI H21-ED6 guideline, supporting the results of the present study. However, this study confirmed that the acceptable room-temperature and refrigerated storage times for FV activity are the same as the acceptable storage times for other coagulation factor and natural anticoagulant activities, excluding FVIII activity. However, FV activity after storage at room temperature for 5 hours decreased the most among coagulation factor and natural anticoagulant activities, excluding FVIII activity. FV activity at room temperature tended to decrease. FV activity has long been thought to be unstable with respect to storage time and temperature in clinical laboratory tests. A recent study using other manufacturers\u0026rsquo; reagents and equipment demonstrated that the acceptable storage time for FV activity at room temperature is shorter than that of other coagulation factors (FII, FVII, FX, FIX, FXI, and FXII) [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe mean percentage change in FVIII activity after storage for 5 hours at room temperature in this study was \u0026minus;\u0026thinsp;43%, indicating that the activity decreased by almost half. Further studies are necessary for samples with originally low activity, such as hemophilia samples. These results suggest that samples from patients with hemophilia (especially for the diagnostic testing of mild hemophilia) pose a risk of misdiagnosis, which could impact patient management.\u003c/p\u003e \u003cp\u003eNext, this study found that storing plasma frozen at -15 to -25\u0026deg;C is not suitable for the stability of FV and FVIII activities. Previous studies using other manufacturers\u0026rsquo; reagents have suggested that FVIII and FIX activities decreased when plasma is stored frozen at -20\u0026deg;C [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. FIX activity was stable within the timeframe of this study, whereas FV and FVIII activities were more unstable than those of the other coagulation factors and natural anticoagulants. Although further studies, such as those including larger sample sizes, using patient samples, or using other manufacturers\u0026rsquo; reagents, are necessary, based on the results of previous studies and this study, caution must be exercised when storing samples for FV, FVIII, and FIX activity testing at freezing temperatures.\u003c/p\u003e \u003cp\u003eFor FV and FVIII activities, which were not stable when plasma was frozen at -15 to -25\u0026deg;C, we changed the storage temperature from freezing at -15 to -25\u0026deg;C to freezing at -75 to -85\u0026deg;C, and we obtained additional data. From the results shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, FV and FVIII activities after frozen storage at -75 to -85\u0026deg;C were stable within the time frame of this study. While the CLSI H21-ED6 guideline does not mention this, the present study recommends storing samples in a deep freezer for measuring FV and FVIII activities when the sample is stored frozen.\u003c/p\u003e \u003cp\u003eIn terms of long-term storage, our results differ from those of a previous study, which reported that FV and FVIII activities were stable for 6 and 3 months, respectively, after frozen storage at -24\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. However, the previous study involved samples collected via plasmapheresis, which differs from the sample collection method used in the present study. The blood collection method used in this study (brachial vein blood collection) was similar to that commonly used in routine clinical laboratory tests. The differences in blood collection methods are assumed to have influenced the results. Although further studies on the stability of FV and FVIII activities are required, long-term storage is not recommended. To obtain appropriate data, measurements must be performed within a short storage period after blood collection.\u003c/p\u003e \u003cp\u003eMoreover, new insights into the effects of storage time and temperature on coagulation factors and natural anticoagulants were obtained for FXIII (room temperature, refrigerated, and frozen), AT (room temperature and refrigerated), PC (room temperature and refrigerated), and free PS (room temperature, refrigerated, and frozen), which have not been previously reported. The coagulation factors and natural anticoagulants remained stable within these timeframes under all storage conditions.\u003c/p\u003e \u003cp\u003eThis study found that the acceptable storage time and temperature varied for each coagulation factor and natural anticoagulant. To provide appropriate measurement data in clinical practice, storage times and temperatures should be critically controlled.\u003c/p\u003e \u003cp\u003eIn summary, this study showed that samples for the measurement of FII, FV, FVII, FX, FIX, FXI, FXII, FXIII, AT, PC, and free PS activities can be stored for up to 5 hours at room temperature or under refrigeration, whereas samples for the measurement of FVIII activity can be stored for only 3 and 4 hours, respectively, at the same temperatures. Also, our findings indicate that samples for the measurement of FII, FVII, FX, FIX, FXI, FXII, FXIII, AT, PC, and free PS activities could be stored frozen at -15 to -25\u0026deg;C for up to 4 months. FV and FVIII activities required frozen storage at -75 to -85\u0026deg;C for the same duration. Nonetheless, this study used plasma samples obtained from only a limited number of healthy individuals. Furthermore, the stability of coagulation factors and natural anticoagulant activities may be affected by patients' clinical states or medications. Therefore, the conclusions of this study should be substantiated by further studies involving different study populations.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eEthics statement\u003c/h2\u003e \u003cp\u003eThis study was approved by the Sysmex Corporation Ethics Committee (approval No., 2021-24). All methods were performed in accordance with Ethical Guidelines for Medical and Biological Research Involving Human Subjects. The internal volunteers provided written informed consent for the use of their samples in this study.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eParticipating centers\u003c/h3\u003e\n\u003cp\u003eThis study was conducted at Sysmex Corporation (Kobe, Japan). Samples were measured using AUTOMATED BLOOD COAGULATION ANALYZER CS-5100 (Sysmex Corp., Kobe, Japan) and Automated Blood Coagulation Analyzer CN-6000 (Sysmex Corp., Kobe, Japan) with Sysmex (Sysmex Corp., Kobe, Japan) and HYPHEN reagents (HYPHEN BioMed, Paris, France).\u003c/p\u003e\n\u003ch3\u003ePlasma samples\u003c/h3\u003e\n\u003cp\u003eHealthy individuals (n\u0026thinsp;=\u0026thinsp;78) were recruited from internal volunteers at the Sysmex Corporation for coagulation factors (FII, FV, FVII, FX, FVIII, FIX, FXI, FXII, and FXIII) and natural anticoagulants (AT, PC, and free PS). Plasma samples were prepared according to the consensus established by Ieko et al. [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. From each volunteer, 9 mL (4.5 mL x 2) blood samples were collected in Venoject II\u0026reg; tubes (Terumo Corp, Tokyo, Japan) containing one-tenth volume of 0.105 M buffered sodium citrate (approximately 3.2%). Whole blood was centrifuged at 1500 \u0026times; \u003cem\u003eg\u003c/em\u003e for 15 minutes at room temperature. The plasma obtained was collected using plastic pipettes and transferred to polystyrene tubes, which were then capped. For each coagulation factor and natural anticoagulant, one group of samples (n\u0026thinsp;=\u0026thinsp;3) was divided between room temperature (18 to 25\u0026deg;C) and refrigerated storage (2 to 8\u0026deg;C), while the other group (n\u0026thinsp;=\u0026thinsp;3) was frozen (-15 to -25\u0026deg;C).\u003c/p\u003e \u003cp\u003eA portion of each sample was analyzed, and the results served as the baseline (0 hours or 0 months). The other samples were tested under the following conditions from baseline measurement: after storage for 2, 3, 4, and 5 hours at room temperature and under refrigeration and for 1, 2, 3, and 4 months at frozen (-15 to -25\u0026deg;C). The refrigerators and freezers used to store the samples were monitored. The samples stored frozen at -15 to -25\u0026deg;C were thawed in a water bath for 15 minutes at 37\u0026deg;C before use.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAssays\u003c/h2\u003e \u003cp\u003eThe plasma samples were measured using the chromogenic method for FVIII, FXIII, AT, PC, and free PS and the clotting method for FII, FV, FVII, FX, FIX, FXI, and FXII. Measurements were conducted using CS-5100 for AT and CN-6000 for FII, FV, FVII, FX, FVIII, FIX, FXI, FXII, FXIII, PC, and free PS. HYPHEN BioMed reagents included Prothrombin Deficient Plasma, Factor V Deficient Plasma, Factor VII Deficient Plasma, Factor X Deficient Plasma, PT-Phen\u0026trade; LRT, Revohem\u0026trade; FVIII Chromogenic, Factor IX Deficient Plasma, Factor XI Deficient Plasma, Factor XII Deficient Plasma, BIOPHEN\u0026trade; Factor XIII, Revohem\u0026trade; AT, Revohem\u0026trade; Protein C, and LIAPHEN\u0026trade; Free Protein S. Sysmex reagents included Revohem\u0026trade; APTT SLA and Revohem\u0026trade; 0.025 M Calcium Chloride Solution. The results are expressed as percentages.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eStatistical analyses\u003c/h3\u003e\n\u003cp\u003eThe measured coagulation factor and natural anticoagulant activities are reported as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). To assess the stability of these activities, the percentage changes from the baseline values were calculated ([result at storage time X - result at baseline] / result at baseline) and averaged at each time point. A mean percentage change of \u0026le;\u0026thinsp;15% compared to the baseline values was considered indicative of acceptable storage.\u003c/p\u003e \u003cp\u003eWhen the mean percentage change in the coagulation factor activity tested after storage at room temperature or under refrigeration exceeded 15% compared with the baseline values, additional samples (n\u0026thinsp;=\u0026thinsp;3) were collected for the coagulation factor activity test and measured after every 30 minutes for 1\u0026ndash;4.5 hours at the same storage temperature. When the mean percentage change in coagulation factor activity after frozen storage (-15 to -25\u0026deg;C) exceeded 15% compared with the baseline values, additional samples (n\u0026thinsp;=\u0026thinsp;3) were collected for the coagulation factor activity test and measured after storage for 1, 2, 3, and 4 months at frozen (-75 to -85\u0026deg;C). When the mean percentage change in the additionally measured results exceeded 15% compared with the baseline values, the measurement was considered complete. The freezers were monitored while storing the samples. The samples stored frozen at -75 to -85\u0026deg;C were thawed in a water bath for 15 minutes at 37\u0026deg;C before use.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003cp\u003eR.N., T.S., D.M., M.K., M.M. and H.M. are employees of Sysmex Corporation. C.D. is an employee of HYPHEN BioMed. Y.K. is an outside adviser for Sysmex Corporation.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eR.N. designed and performed the experiments, analyzed the data, and drafted the manuscript. D.M., M.K., M.M., and C.D. designed the experiments. Y.K. interpreted the data and revised the manuscript. T.S. and H.M. supervised the study and revised the manuscript. All the authors critically reviewed and approved the final version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThis study was supported by the Sysmex Corporation.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets analyzed during this study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGosselin, R. C. \u0026amp; Marlar, R. A. Preanalytical variables in coagulation testing: setting the stage for accurate results. \u003cem\u003eSemin. Thromb. Hemost.\u003c/em\u003e \u003cstrong\u003e45\u003c/strong\u003e, 433-448 (2019).\u003c/li\u003e\n\u003cli\u003eAdcock, D. M., Favaloro, E. J. \u0026amp; Lippi, G. Critical pre-examination variables in the hemostasis laboratory and their quality indicators. \u003cem\u003eClin. Biochem.\u003c/em\u003e \u003cstrong\u003e49\u003c/strong\u003e, 1315\u0026ndash;1320 (2016).\u003c/li\u003e\n\u003cli\u003eFavaloro, E. J., Lippi, G. \u0026amp; Adcock, D. M. Preanalytical and postanalytical variables: the leading causes of diagnostic error in hemostasis? \u003cem\u003eSemin. Thromb. Hemost.\u003c/em\u003e \u003cstrong\u003e34\u003c/strong\u003e, 612-634 (2008).\u003c/li\u003e\n\u003cli\u003eCLSI. Collection, Transport, and Processing of Blood Specimens for Testing Plasma-Based Coagulation Assays- \u003cem\u003e6th ed. CLSI document\u003c/em\u003e H21-ED6 Wayne (Clinical and Lab. Standards Inst., PA, 2024). \u003c/li\u003e\n\u003cli\u003eFeng, L., Zhao, Y., Zhao, H. \u0026amp; Shao, Z. Effects of storage time and temperature on coagulation tests and factors in fresh plasma. \u003cem\u003eSci. Rep.\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, 3868 (2014). \u003c/li\u003e\n\u003cli\u003eZhao, Y., Feng, G. \u0026amp; Feng, L. Effects of pre-analytical storage time, temperature, and freeze-thaw times on coagulation factors activities in citrate-anticoagulated plasma. \u003cem\u003eAnn. Transl. Med. \u003c/em\u003e\u003cstrong\u003e6\u003c/strong\u003e, 456 (2018).\u003c/li\u003e\n\u003cli\u003eB\u0026ouml;hm, M. \u003cem\u003eet al\u003c/em\u003e. Cold storage of citrated whole blood induces drastic time-dependent losses in factor VIII and von Willebrand factor: potential for misdiagnosis of haemophilia and von Willebrand disease. \u003cem\u003eBlood Coagul. Fibrinolysis\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, 39-45 (2006). \u003c/li\u003e\n\u003cli\u003eLinskens, E. A. \u0026amp; Devreese, K. M. J. Pre-analytical stability of coagulation parameters in plasma stored at room temperature. \u003cem\u003eInt. J. Lab. Hematol.\u003c/em\u003e \u003cstrong\u003e40\u003c/strong\u003e, 292-303 (2018).\u003c/li\u003e\n\u003cli\u003eZhao, Y. \u003cem\u003eet al\u003c/em\u003e. Effects of preanalytical frozen storage time and temperature on screening coagulation tests and factors VIII and IX activity. \u003cem\u003eSci. Rep. \u003c/em\u003e\u003cstrong\u003e7\u003c/strong\u003e, 12179 (2017).\u003c/li\u003e\n\u003cli\u003eWoodhams, B., Girardot, O., Blanco, M. J., Colesse, G. \u0026amp; Gourmelin, Y. Stability of coagulation proteins in frozen plasma. \u003cem\u003eBlood Coagul. Fibrinolysis \u003c/em\u003e\u003cstrong\u003e12\u003c/strong\u003e, 229-236 (2001).\u003c/li\u003e\n\u003cli\u003eIeko, M. \u003cem\u003eet al\u003c/em\u003e. Expert consensus regarding standardization of sample preparation for clotting time assays. \u003cem\u003eInt. J. Hematol. \u003c/em\u003e\u003cstrong\u003e112,\u003c/strong\u003e 614-620 (2020).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"pre-analytical variable, coagulation tests, stability, storage conditions, Clinical and Laboratory Standards Institute","lastPublishedDoi":"10.21203/rs.3.rs-5314705/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5314705/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCoagulation test results are influenced by many pre-analytical variables, including clinical sample storage conditions. This study investigated the effects of storage time and temperature on coagulation factor (FII, FV, FVII, FX, FVIII, FIX, FXI, FXII, and FXIII) and natural anticoagulant (antithrombin, protein C, and free protein S) activities. Blood samples from healthy individuals were centrifuged and plasma was tested after room temperature (18 to 25\u0026deg;C) or refrigerated (2 to 8\u0026deg;C) for 2, 3, 4, and 5 hours, or after frozen storage (-15 to -25\u0026deg;C) for 1, 2, 3, and 4 months. For all storage conditions, the mean percentage change from baseline in evaluated activities was \u0026lt;\u0026thinsp;15%, except for FV and FVIII. For FV activities after frozen storage at -15 to -25\u0026deg;C and FVIII activities at all storage conditions, the mean percentage change was \u0026gt;\u0026thinsp;15%. For FV and FVIII activities, the mean percentage change was \u0026lt;\u0026thinsp;15% after frozen storage at -75 to -85\u0026deg;C for 1, 2, 3, and 4 months. These labile factors are associated with rapid decreases in plasma levels \u003cem\u003ein vitro\u003c/em\u003e. To ensure appropriate clinical testing results, especially for FV and FVIII activity, storage condition and time should be critically controlled.\u003c/p\u003e","manuscriptTitle":"Effects of storage time and temperature on coagulation factor and natural anticoagulant activities","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-25 14:09:35","doi":"10.21203/rs.3.rs-5314705/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-12-05T09:13:53+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-12-05T01:36:43+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-29T18:23:27+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-27T11:57:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"58774977531414663156765697729177851520","date":"2024-11-19T13:53:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"13999564556753110703739953186839499092","date":"2024-11-18T14:15:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"268439199242011619754795468310055689495","date":"2024-11-18T14:00:02+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-11-18T13:56:27+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-11-18T12:10:19+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-11-07T07:05:11+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-11-07T05:15:44+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-10-23T00:51:07+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e7924baa-0fd2-41f7-bb5a-1d2e8500135b","owner":[],"postedDate":"November 25th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":40471904,"name":"Biological sciences/Immunology/Coagulation system"},{"id":40471905,"name":"Biological sciences/Biotechnology/Assay systems"},{"id":40471906,"name":"Biological sciences/Biological techniques/Analytical biochemistry/Biochemical assays"},{"id":40471907,"name":"Biological sciences/Drug discovery/Diagnostics"},{"id":40471908,"name":"Health sciences/Health care/Diagnosis/Laboratory techniques and procedures"},{"id":40471909,"name":"Physical sciences/Chemistry/Analytical chemistry/Medical and clinical diagnostics"}],"tags":[],"updatedAt":"2025-03-31T16:04:28+00:00","versionOfRecord":{"articleIdentity":"rs-5314705","link":"https://doi.org/10.1038/s41598-025-95389-w","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-03-28 15:57:16","publishedOnDateReadable":"March 28th, 2025"},"versionCreatedAt":"2024-11-25 14:09:35","video":"","vorDoi":"10.1038/s41598-025-95389-w","vorDoiUrl":"https://doi.org/10.1038/s41598-025-95389-w","workflowStages":[]},"version":"v1","identity":"rs-5314705","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5314705","identity":"rs-5314705","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}