兽研缩写
新鲜冰冻血浆
凝血酶原时间
部分凝血活酶时间
浓缩红细胞
血管性血友病因子
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引言
新鲜冰冻血浆（FFP）是一种宝贵的血库资源，对于治疗伴有凝血功能障碍、出血、低蛋白血症和内皮糖萼功能障碍等情况的危重患者至关重要[1, 2]。为符合血液与生物治疗促进协会制定的标准，FFP需在采集后规定时间内从全血中分离，并可储存于−18°C或更冷的冰柜中[3]。按照这些指南储存时，FFP可维持所有功能性止血蛋白、白蛋白和球蛋白的治疗浓度[4, 5]。
为准备输血，血浆应在适当的血浆解冻装置或水浴中（温度介于30°C至37°C之间）解冻，然后立即输注或置于1°C–6°C冰箱中保存，使用前不超过5天[3]。另外，一项兽医研究表明，犬和猫FFP单位在经历至少一次冻融循环后均能保持止血能力[6]。更近期的文献提示，解冻后的犬FFP在冷藏或室温下延长储存后，仍能维持凝血因子（包括热不稳定因子V和VIII）的治疗浓度[7, 8]。尚未有猫用产品的类似发现发表。本报告的目的是描述在血库冰柜意外故障导致冰柜内物品暴露于室温长达10小时后，代表性犬和猫FFP单位的止血能力并评估细菌生长情况。
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材料与方法
约11单位犬FFP、7单位猫FFP和5单位猫冰冻血浆储存于某大学教学医院的商用血库冰柜1中供临床使用。所有单位均按照机构方案采用无菌技术采集和处理[9]。血浆单位储存于−30°C，直至冰柜门意外整夜未关，次日早晨发现这些单位完全呈液态，时间长达10小时。经逐个检查和处理，每个单位主观上已与室温（20°C）平衡。从解冻血浆池中随机选取位于各自抽屉中央的一个犬FFP单位（来源于院内献血计划）和一个猫FFP单位（来源于外部兽医血库2）进行检测。使用检查手套和无菌技术，将带有无针接头的无菌静脉输液袋穿刺器插入血袋的一个穿刺口。用异丙醇清洁鲁尔接头表面并待干。使用无菌注射器从单位中抽取血浆样本用于凝血检测和培养。凝血酶原时间（PT）和部分凝血活酶时间（PTT）使用院内分析仪3测定。每个单位的细菌培养在层流罩下进行，将血浆接种至需氧和厌氧培养基4中，然后在受控条件下5孵育5天。使用新的无菌针头向每个培养瓶接种5 mL血浆。从每个单位另收集10 mL血浆，以0.5 mL等分试样重新冷冻于−30°C。这些样本被送至外部实验室6进行因子XIII缺乏筛查以及纤维蛋白原、因子II、V、VII、VIII、IX、X、XI和XII的凝血活性及血管性血友病因子（vWF）的测定。
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结果
犬和猫血浆样本的PT和PTT时间均正常（表1）。两个血浆单位的因子XIII缺乏筛查均正常，表明因子XIII活性至少为5%。所有凝血因子活性均在参考区间内或超过参考区间（表2）。在猫样本中，活性最低的因子为VIII（76%）和vWF（经胶原结合试验评估为77%）。犬血浆样本的所有因子分析结果均超过100%。两个单位孵育5天后细菌培养均为阴性。
TABLE 1. Results of prothrombin time (PT) and partial thromboplastin time (PTT) from representative samples of canine and feline plasma after exposure to room temperature for approximately 10 h. All values in seconds.
| Canine plasma sample | Canine reference interval | Feline plasma sample | Feline reference interval | |
|---|---|---|---|---|
| PT | 7.9 | 7–9.4 | 11.9 | 8.7–12.6 |
| PTT | 12.3 | 8.5–13.8 | 16.2 | 8.9–17.4 |
TABLE 2. Coagulation factor analysis results in representative samples of canine and feline plasma after exposure to room temperature for approximately 10 h.
| Analyte | Canine factor activity | Feline factor activity | Reference interval (canine and feline) |
|---|---|---|---|
| Factor II | 137% | 127% | 50%–150% |
| Factor V | 122% | 157% | 50%–150% |
| Factor VII | 161% | 102% | 50%–150% |
| Factor VIII | 175% | 76% | 50%–200% |
| Factor IX | 177% | 102% | 50%–150% |
| Factor X | 123% | 104% | 80%–175% |
| Factor XI | 211% | 165% | 60%–150% |
| Factor XII | 205% | 119% | 60%–150% |
| Fibrinogen | 304 mg/dL | 401 mg/dL | 76–270 mg/dL |
| vWF (vWF:Ag) | 149% | 97% | 70%–180% |
| vWF (vWF:CBA) | 134% | 77% | 50%–170% |
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讨论
本报告描述了在大学血库冷冻柜中储存的犬和猫血液制品意外暴露于室温条件下长达10小时后所进行的调查。对代表性血浆样本进行了凝血因子活性检测，并验证在这些条件下未发生细菌生长。作者进行这些检测是为了评估剩余血浆制品用于物种特异性临床患者输注的安全性和实用性，而非将剩余产品降级或丢弃。
本报告中的发现与近期一项离体研究结果一致，该研究证明20单位犬新鲜冷冻血浆在模拟恒速输注期间室温放置12小时后，因子V、VII、VIII和IX保持稳定，且无细菌生长[7]。同样，此处检测的犬和猫制品均显示出可接受的凝血因子活性和vWF浓度。两种产品均未显示细菌生长。
人医学中血浆产品的一般质量保证指南是1 mL新鲜冷冻血浆约含1单位凝血因子活性。这一标准已被兽医学采纳，其中各因子活性以百分比值报告，代表每毫升活性单位数（U/mL）。这与混合犬血浆标准品进行比较，后者赋值为1 U/mL或100%[5]。欧洲新鲜冷冻血浆质量控制标准基于不稳定因子VIII活性不低于0.7 U/mL或70%[10]。同样，在兽医学中，犬和猫新鲜冷冻血浆在面临温度变化或延长储存时，因子VIII会发生显著降解[4, 5]。除因子VIII外，猫新鲜冷冻血浆储存1年后活性最低的因子为VII和XII[4]，而犬新鲜冷冻血浆储存1年后活性最低的因子为IX和X[5]。在一项犬新鲜冷冻血浆在模拟恒速输注期间室温放置12小时的研究中，作者未证明暴露于室温4或12小时后因子V、VII、VIII或IX活性降低[7]。在本研究提供的数据中，因子VIII及所有其他因子活性均在可接受范围内，仅猫因子VIII活性低于100%。各物种的PT和PTT检测均保持在正常范围内，提示对于治疗凝血病具有治疗充分性。
本报告为围绕血液制品输注“4小时规则”有效性的日益增多的证据增添了新内容。这一人医学中的建议源于输血相关脓毒症并发症，更典型地与血小板制品相关，因为其制备时使用含更多能量底物的添加剂溶液，且储存温度高于浓缩红细胞（pRBCs）或血浆[3, 11]。然而，这一“规则”已被外推至包括浓缩红细胞、新鲜冷冻血浆及其他人医学和兽医学中的成分输血治疗。在一项关于浓缩红细胞暴露时间的系统综述中，作者报告648单位中有5单位（0.8%）在室温暴露约6小时后出现细菌生长，而六项评估研究中四项未在任何检测单位中发现细菌生长[12]。在所综述的研究中，暴露时间和温度暴露程度从数小时到长达1天不等，使得难以就特定安全或不安全时间范围得出有意义的结论。此外，证据质量和研究年代也构成局限，因为血液采集和储存技术已随时间发展。这些局限性使作者无法做出强有力的临床建议，尽管他们提出浓缩红细胞的“4小时规则”可延长至5小时或更长而不显著损害血液质量[12]。然而，鉴于输注可能被污染的浓缩红细胞具有严重的临床后果，在记录更稳健的结论之前，应谨慎解读这一评论。兽医学文献中数据很少，但近期一项多机构监测研究报告称，19%的犬浓缩红细胞输注超过了4小时时限，且任何患者均无输血相关脓毒症证据[13]。Mesa-Sanchez等人评估了犬新鲜冷冻血浆在室温暴露4和12小时后细菌生长的证据，未发现污染证据[7]。我们的报告首次证明猫新鲜冷冻血浆制品在类似时间室温暴露后的无菌性。
本研究的局限性包括：所分析的血袋缺乏基线凝血因子活性数据，导致无法量化凝血因子活性的下降幅度，且受影响血袋的确切暴露时间未知。解冻速率可能因 freezer 门未关严的程度及血袋在 freezer 内的具体位置而偏快或偏慢，进而可能影响产品降解程度。研究对代表性血袋进行了检测，而非破坏所有血袋的完整性；然而，若对所有血袋进行检测，个体血袋间的结果可能存在差异。这些局限性使得难以将这些数据外推至临床使用场景，例如在超过4小时的时间范围内输注FFP时。
总之，两份犬和猫血袋在室温条件下暴露长达10小时后，凝血因子活性和无菌性均得以维持。对代表性血袋进行类似分析可能对可能遭遇 freezer 故障的血库或医院有用，可提示产品可能无需降级或丢弃。需要更大规模的对照研究来支持这些结果。
作者贡献
Samantha J. Bilko：概念构思、研究实施、写作——审阅与编辑、写作——初稿。Julie M. Walker：概念构思、研究实施、写作——审阅与编辑、监督指导。
伦理声明
作者确认已遵守期刊作者指南页面所载的期刊伦理政策。本研究未使用活体动物，故无需伦理批准。
利益冲突
作者声明无利益冲突。
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Abbreviations
FFPfresh frozen plasma
PTprothrombin time
PTTpartial thromboplastin time
pRBCpacked red blood cell
vWFvon Willebrand factor
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Introduction
Fresh frozen plasma (FFP), a valuable blood bank resource, is essential in the treatment of critically ill patients with conditions including coagulopathy, hemorrhage, hypoproteinemia, and endothelial glycocalyx dysfunction [1, 2]. To meet standards set by the Association for the Advancement of Blood and Biotherapies, FFP is separated from whole blood within a defined time period from collection and can be stored in a freezer −18°C or colder [3]. When stored according to these guidelines, FFP maintains therapeutic concentrations of all functional hemostatic proteins, albumin, and globulins [4, 5].
To prepare for transfusion, plasma should be thawed in an appropriate plasma thawing device or water bath (temperature between 30°C and 37°C), then transfused immediately or kept in a refrigerator at 1°C–6°C for no more than 5 days before use [3]. Alternatively, one veterinary study demonstrated that both canine and feline FFP units maintain hemostatic capacity after sustaining at least one freeze–thaw cycle [6]. More recent literature suggests that thawed canine FFP maintains therapeutic concentrations of clotting factors, including heat‐labile factors V and VIII, through prolonged storage under refrigeration or at room temperatures [7, 8]. Similar findings have not been published for feline product. The purpose of this report is to describe the hemostatic capacity and assess for bacterial growth in representative units of canine and feline FFP after an inadvertent freezer malfunction led to the contents of a blood bank freezer being exposed to room temperature for up to 10 h.
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Materials and Methods
Approximately 11 units of canine FFP, seven units of feline FFP, and five units of feline frozen plasma were stored for clinical use in a commercial blood bank freezer1 at a university teaching hospital. All units were collected and processed using aseptic technique according to institutional protocols [9]. The plasma units were stored at −30°C until the freezer door was inadvertently left open overnight and the units were found in a completely liquid state the following morning, up to 10 h later. Upon individual examination and handling, each unit had subjectively equilibrated with the temperature of the room (20°C). One canine FFP unit (sourced from the in‐house blood donor program) and one feline FFP unit (sourced from an outside veterinary blood bank2) located centrally within their respective drawers were randomly selected from the thawed plasma pool for testing. Using examination gloves and aseptic technique, a sterile intravenous bag spike with a needleless connector was inserted into one of the bag's spike ports. The Luer adapter surface was cleaned with isopropyl alcohol and allowed to dry. A sterile syringe was used to withdraw a sample of plasma from the unit for coagulation testing and culture. Prothrombin time (PT) and partial thromboplastin time (PTT) were measured using an in‐house analyzer3. Bacterial culture was performed under a laminar flow hood on each unit by inoculating plasma into aerobic and anaerobic culture media4, which were then incubated under controlled conditions5 for 5 days. A new, sterile needle was used to inoculate each vial with 5 mL of plasma. An additional 10 mL of plasma was collected from each unit and refrozen in 0.5‐mL aliquots at −30°C. These were submitted for factor XIII deficiency screening and measurement of fibrinogen, coagulant activity of factors II, V, VII, VIII, IX, X, XI, and XII, and von Willebrand factor (vWF) at an outside laboratory6.
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Results
Both canine and feline plasma samples had normal PT and PTT times (Table 1). Factor XIII deficiency screening was normal for both plasma units, indicating at least 5% factor XIII activity. All coagulant factor activities remained within or exceeded the reference intervals (Table 2). In the feline sample, the factors with the lowest activities were VIII (76%) and vWF (77% as assessed via collagen binding assay). Results of all factor analyses exceeded 100% in the canine plasma sample. Bacterial cultures for both units were negative after incubation for 5 days.
TABLE 1. Results of prothrombin time (PT) and partial thromboplastin time (PTT) from representative samples of canine and feline plasma after exposure to room temperature for approximately 10 h. All values in seconds.
| Canine plasma sample | Canine reference interval | Feline plasma sample | Feline reference interval | |
|---|---|---|---|---|
| PT | 7.9 | 7–9.4 | 11.9 | 8.7–12.6 |
| PTT | 12.3 | 8.5–13.8 | 16.2 | 8.9–17.4 |
TABLE 2. Coagulation factor analysis results in representative samples of canine and feline plasma after exposure to room temperature for approximately 10 h.
| Analyte | Canine factor activity | Feline factor activity | Reference interval (canine and feline) |
|---|---|---|---|
| Factor II | 137% | 127% | 50%–150% |
| Factor V | 122% | 157% | 50%–150% |
| Factor VII | 161% | 102% | 50%–150% |
| Factor VIII | 175% | 76% | 50%–200% |
| Factor IX | 177% | 102% | 50%–150% |
| Factor X | 123% | 104% | 80%–175% |
| Factor XI | 211% | 165% | 60%–150% |
| Factor XII | 205% | 119% | 60%–150% |
| Fibrinogen | 304 mg/dL | 401 mg/dL | 76–270 mg/dL |
| vWF (vWF:Ag) | 149% | 97% | 70%–180% |
| vWF (vWF:CBA) | 134% | 77% | 50%–170% |
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Discussion
This report describes the investigation that was performed after canine and feline units stored in a university blood bank freezer were accidentally exposed to room temperature conditions for up to 10 h. Representative plasma samples were tested for coagulant factor activity and to verify that bacterial growth had not occurred under these conditions. The authors performed these tests to assess the safety and utility of the remaining plasma units for species‐specific administration to clinical patients, rather than downgrading or discarding the remaining product.
The findings presented in the current report are consistent with the results of a recent ex vivo study that demonstrated stability of factors V, VII, VIII, and IX, and lack of bacterial growth when 20 units of canine FFP were held at room temperature for 12 h during simulated constant rate infusion [7]. Similarly, both canine and feline units tested here demonstrated acceptable coagulation factor activities and vWF concentration. Neither product demonstrated bacterial growth.
A general quality assurance guideline for plasma products in human medicine is that 1 mL of FFP contains approximately 1 unit of coagulation factor activity. This has been adopted in veterinary medicine, where individual factor activity is reported as a percentage value, representing the units of activity per milliliter (U/mL). This is compared with a pooled canine plasma standard, which has an assigned value of 1 U/mL, or 100% [5]. A European standard for FFP quality control is based on labile factor VIII activity of no less than 0.7 U/mL, or 70% [10]. Similarly, in veterinary medicine, factor VIII undergoes significant degradation in canine and feline FFP when faced with temperature variability or prolonged storage [4, 5]. Other than factor VIII, the factors demonstrating the lowest activities in feline FFP after 1 year of storage were VII and XII [4], while the factors with the lowest activities in stored canine FFP after 1 year were IX and X [5]. In a study of canine FFP held at room temperature for 12 h during simulated constant rate infusion, the authors did not demonstrate a reduction in factor V, VII, VIII, or IX activity after 4 or 12 h of exposure to room temperature [7]. In the data presented in this study, factor VIII and all other factor activities were within the acceptable range, with only feline factor VIII demonstrating activity below 100%. PT and PTT testing remained within normal range for each species, suggesting therapeutic adequacy for treating coagulopathy.
The current report adds to the growing body of evidence surrounding the validity of the “4‐h rule” for transfusing blood products. This recommendation in human medicine originates from transfusion‐associated sepsis complications, more typically associated with platelet products due to their preparation with additive solutions containing more energy substrates and storage at higher temperatures than packed red blood cells (pRBCs) or plasma [3, 11]. However, this “rule” has been extrapolated to include pRBCs, FFP, and other component therapy in both human and veterinary medicine. In a systematic review of exposure times in pRBC units, the authors reported bacterial growth in five of 648 (0.8%) units that sustained room temperature exposure for approximately 6 h, with four of six evaluated studies finding no bacterial growth in any tested units [12]. Among the studies reviewed, the length of time and degree of temperature exposure varied from hours to up to 1 day, making it difficult to draw meaningful conclusions about a specific safe or unsafe time frame. Additionally, the quality of evidence and the age of the studies were limiting, as blood collection and storage techniques have evolved over time. These limitations left the authors unable to make a strong clinical recommendation, although they offered a suggestion that the “4‐h rule” for pRBCs could be extended to 5 h or longer without significant detriment to blood quality [12]. However, given the serious clinical implications of transfusing pRBCs that could be contaminated, this comment should be interpreted cautiously until more robust conclusions can be documented. Few data exist within the veterinary literature, but a recent multi‐institutional surveillance study reports that 19% of canine pRBC transfusions administered exceeded the 4‐h time limit with no evidence of transfusion‐associated sepsis in any patient [13]. Mesa‐Sanchez et al. evaluated canine FFP for evidence of bacterial growth after 4 and 12 h of exposure to room temperature and found no evidence of contamination [7]. Our report is the first to demonstrate the sterility of a feline FFP unit after being exposed to room temperature for a similar period of time.
Limitations to this investigation include the absence of baseline coagulation factor activity in the analyzed units, making it impossible to quantify the decline in clotting factor activity, as well as the precise timing of exposure of the affected units being unknown. The rate of thaw may have been faster or slower depending on the extent to which the freezer door was left ajar and the exact placement of units within the freezer, which in turn may have affected the degree of product degradation. Representative units were tested, rather than compromising the integrity of all units; however, it is possible that results may have differed among individual units, had they all been tested. These limitations make it difficult to extrapolate these data to clinical use, such as when administering FFP infusions over a time frame greater than 4 h.
In conclusion, coagulation factor activity and sterility were maintained in two canine and feline units after exposure to room temperature conditions for up to 10 h. Performing similar analyses on representative units might be useful to blood banks or hospitals, which may experience freezer malfunction, to indicate that product may not need to be downgraded or discarded. A larger controlled study is warranted to support these results.
Author Contributions
Samantha J. Bilko: conceptualization, investigation, writing – review and editing, writing – original draft. Julie M. Walker: conceptualization, investigation, writing – review and editing, supervision.
Ethics Statement
The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to. No ethical approval was required as no live animals were used in this study.
Conflicts of Interest
The authors declare no conflicts of interest.