Stability of prothrombin time and activated partial thromboplastin time tests under different storage conditions

Prothrombin time (PT) and activated partial thromboplastin time (aPTT) are common laboratory tests that are useful in the diagnosis of coagulation disorders and monitoring anticoagulant therapy. Recent expansions in the outreach laboratory services at our institution prompted us to investigate the shipping limitations for some tests, including PT and aPTT. Although we followed NCCLS guidelines for the collection of blood specimens, we observed falsely elevated PT and aPTT values due to the different storage conditions. The objective of this study is to determine the effect of conditions and duration of storage on PT and aPTT tests using plasma and whole blood samples, respectively. For this study, 36 plasma samples with normal and prolonged PT and aPTT were exposed to different storage conditions. Blood was centrifuged immediately and plasma was stored at room temperature (RT), refrigerated at 4°C, or frozen at −20°C. The samples were analyzed at 0 h and repeated at 6, 12 and 24 h under various conditions. Although statistically significant differences were observed for plasma samples for normal PT tests after 12 h at refrigerated and frozen storage conditions, the differences would not change the clinical interpretation of the results. On the other hand, samples stored refrigerated or at RT showed significant differences for aPTT at 24 h. These differences would change clinical interpretation, especially for samples with normal or near normal aPTT times. Interestingly, aPTT was significantly higher for samples stored frozen when compared to refrigerated and RT conditions at 6 h. Similar patterns were also observed on ten whole blood samples with normal PT and aPTT values. In conclusion, either plasma or whole blood samples can be accepted for PT testing up to 24 h and for aPTT testing up to 12 h only, when transported either at RT or at 4°C.     

Preferential consumption of coagulation factors I, V, and VIII in rat endotoxemia

To ascertain the time course of prolonged coagulation time and the coagulation factors that were consumed preferentially after injection of Escherichia coli endotoxin (ETX, 3 mg/kg, intravenously) in rats, the activated partial thromboplastin time (aPTT) and prothrombin time (PT) were measured. Using aPTT and PT, the residual levels of the major coagulation factors were quantified by partial replacement of ETX-injected rat plasma with individual factor-deficient human plasma. The residual levels of prekallikrein and high molecular weight (HMW) kininogen were also measured. After ETX injection, aPTT and PT showed gradual increasing prolongation, which was marked at 3-5 h after the injection. The residual level of fibrinogen was markedly reduced between 1 and 3 h after ETX injection and dropped to the determination limit 7 h after the injection. Ratios of the consumed coagulation factors, prekallikrein, and HMW kininogen in rat plasma collected 7 h after intravenous injection of ETX were obtained as follows: prekallikrein (18.0 +/- 4.8%), HMW kininogen (36.2 +/- 1.9 %), factor XII (54.0 +/- 0.7%), factor VIII (86.1 +/- 1.8%), factor VII (35.6 +/- 7.7%), factor V (90.6 +/- 0.8%), and factor I (fibrinogen) (>89.6 +/- 0.0%). Thus, coagulation factor I (fibrinogen) and factors V and VIII (cofactors) were consumed preferentially. The extrinsic coagulation pathway was dominantly activated, whereas the intrinsic coagulation pathway, including plasma kallikrein-kinin system, played less important role in the ETX-induced consumption coagulopathy in rat.     

Refreezing previously thawed fresh-frozen plasma. Stability of coagulation factors V and VIII:C

With the growth in autologous blood programs and the increased scrutiny of the indications for transfusion of fresh-frozen plasma (FFP), an increase has been seen in the number of occasions on which FFP was requested and thawed but then not transfused. The coagulation properties of FFP units that were refrozen and then rethawed were therefore studied. Fifty-eight units of plasma were studied, with each experimental unit of FFP paired with an identical control unit. Experimental units were frozen, stored at -65 degrees C, thawed, stored at 1 to 6 degrees C for various periods of time up to 24 hours, and then refrozen, stored at -65 degrees C, rethawed, and stored again in the refrigerator for up to 24 hours. Control units were frozen once at the time the experimental units were first frozen and thawed once at the time of the second thaw of the experimental units. Aliquots of plasma were sampled periodically and were later batch-tested for prothrombin time (PT), activated partial thromboplastin time (aPTT), and factor V and VIII:C activity. The results of coagulation testing of the twice-frozen plasmas were always within the normal range. There was a slight but statistically valid prolongation of the PT and aPTT and a decrease in the factor V and VIII:C levels for twice-frozen plasma compared with control plasma. The greatest decline occurred in the level of factor VIII:C. The measured deterioration in coagulation of twice-frozen FFP is unlikely to be of clinical importance. Refreezing FFP may eventually prove useful for rare donor, autologous, and massive transfusion programs.     

Stability of 5-fluorouracil in whole blood and plasma

We studied the stability of 5-fluorouracil (5-FU) in plasma and whole blood kept at room temperature and on ice for 1 to 24 h. At room temperature, there was a steady loss of 94% of the parent drug over 24 h in whole blood and 52% in plasma. In the presence of an excess of uracil, 5-FU was stable for 24 h, suggesting that the loss of 5-FU is the result of enzymatic degradation. 5-FU is more stable in whole blood and plasma when samples are kept cold. For blood and plasma samples maintained on ice, the loss was only 30% and 10% of the parent drug in the respective samples over 24 h. Frozen plasma samples (-20 degrees C) were stable for five weeks. Blood specimens collected for quantifying 5-FU should be immediately placed on ice, and the plasma should be separated and frozen as promptly as possible.     

Effects of telavancin on coagulation test results

Background: The lipoglycopeptide antibiotic, telavancin, may interfere with some laboratory coagulation tests including prothrombin time (PT) and activated partial thromboplastin time (aPTT). Objective: To evaluate the effects of telavancin on PT and aPTT assays in common use. Methods: Pooled normal human plasma was spiked with telavancin 10, 20, 100 or 200 μg/ml (equivalent to trough, 2 × trough, peak and 2 × peak clinical plasma concentrations, respectively) or diluent control (0.9% sodium chloride). Samples were analysed using 16 PT reagents and seven aPTT reagents. Results: Telavancin 200 μg/ml (corresponding to 2 × peak clinical plasma concentration), produced significant PT prolongation (> 9% difference vs. diluent control) with all the 16 PT reagents (range 12% to > 600%). At lower telavancin concentrations, PT prolongation was dose‐dependent and varied among reagents, but appeared greatest with preparations containing recombinant tissue factor. With telavancin 10 μg/ml (equivalent to trough), PT prolongation was 10% with HemosIL^® PT‐Fibrinogen Recombinant, while ranging from 5% to –1% with all other reagents. Significant (> 34% difference vs. baseline) and dose‐dependent aPTT prolongation was observed with all the seven reagents in samples spiked with telavancin 100 or 200 μg/ml (range 65–142% at 200 μg/ml). aPTT reagents containing a silica activator appeared to be more sensitive to telavancin interference. Telavancin 10 μg/ml was not associated with increased aPTT with any of the reagents tested. Conclusions: Telavancin has the potential to prolong both PT and aPTT in vitro. It is recommended that samples for PT or aPTT be obtained just prior to a telavancin dose (trough).     

新型血管内液体栓塞剂中有效组分的体外促凝血作用研究

目的研究用于颅内动脉瘤治疗的新型血管内液体栓塞剂中有效栓塞成分(白芨多糖与二醋酸纤维素聚合物)的体外促凝血作用。方法将有效栓塞成分按质量比不同分为4组,观察各组样品对凝血酶原时间(PT)与活化部分凝血活酶时间(aPTT)的影响。结果新型血管内液体栓塞剂能显著缩短PT,且缩短程度与主要栓塞成分白芨多糖所占比例成正相关;同时此栓塞剂也可缩短aPTT。结论新型血管内液体栓塞剂在体外实验中明显促进凝血,并偏重于影响外源性凝血途径;此外,主要栓塞成分白芨多糖在促进凝血过程中起关键作用。     

Reference intervals for coagulation tests in adults with different ABO blood types

IntroductionCoagulation tests are affected by many factors, such as age, race, and gestation. Although coagulation test results vary by ABO blood type, reference intervals of different ABO blood groups remain to be determined. This study aims to investigate the reference ranges of coagulation tests for different ABO blood groups in the Han population in South China.MethodsA retrospective study was conducted in the First Affiliated Hospital of Shantou University Medical College. In all, 9600 individuals aged between 20 and 79 years were included. Coagulation tests, including prothrombin time (PT), international normalized ratio (INR), activated partial thromboplastin time (aPTT), thrombin time, and fibrinogen, were performed.ResultsThere was a significant difference in PT, INR, and aPTT among ABO blood groups. PT and INR varied slightly between ABO blood groups. There was a higher aPTT value in individuals in the O blood group than in those in non‐O blood groups, in both males and females across the included age range. No differences were found in thrombin time and fibrinogen between the ABO blood groups.ConclusionThe study provides reference data on coagulation tests from ABO blood groups in South China. The established reference intervals specific to ABO blood type, sex, and age may improve clinical decisions based on coagulation tests.     

洗涤式自体血回输联合新鲜冰冻血浆预防术后凝血功能障碍

【目的】探讨洗涤自体血回输联合使用新鲜冰冻血浆预防术后凝血障碍的疗效。【方法】选取2012年1月至2013年12月本院收治的大出血患者90例,按照患者的术中失血量大小,将其分为小量组（＜400mL,n＝50）、中量组（400～800mL,n＝20）和大量组（＞800mL,n＝20）。根据回输血量多少与患者凝血功能的量化关系,制定联合新鲜冰冻血浆输注的具体方案。比较分析三组患者纤维蛋白原（FIB）、血小板（PLT）及凝血酶原时间（PT）变化情况。【结果】回输血液后三组患者机体FIB、PLT均较术前显著降低,PT明显延长（P＜0．05）;术后18h三组患者FIB、PLT均较回输血液后明显升高,PT显著缩短（P＜0．05）。【结论】联合新鲜冰冻血浆输注的具体方案能提高洗涤自体血回输的安全性和实用性,有效预防凝血功能障碍的发生。     

凝血试验预测口腔颌面外科手术出血的价值

目的:评估术前常规凝血试验(coagulation tests,CTs)预测口腔颌面外科手术出血的价值。方法:对2002年12月- 2003年12月接受口腔颌面外科大手术的387例病人进行回顾性研究,通过术前常规CTs筛选出12例凝血功能异常的病人并匹配24例对照,对两组间出血相关临床事件及手术时间、术中失血和输血量进行分析。结果:凝血功能异常的发病率为3.10%,其中血小板计数(PC)异常的发生率为2.07%,凝血酶原时间(PT)和活化部分凝血活酶时间(aPTT)异常的发生率为1.34%,CTs筛选出的无任何临床症状和体征的凝血功能异常为1.29%(5/387)。病例组和对照组相比,在延缓手术或改变手术方式(66.7%vs.0%,P=0.000016)、术中术后全身使用生物止血剂(58.3%vs.8.3%,P=0.0025)等方面存在显著差异, 病例组平均手术时间明显短于对照组(5.77±1.37h vs.5.84±2.54h,P<0.01);而在术中因失血引起低血压(16.7%vs. 4.2%,P=0.28),因出血再次手术(8.3%vs.4.2%,P=0.56),因出血导致死亡(8.3%vs.0%,P=0.33)及术中失血和输血量无显著差异(P>0.05)。结论:口腔颌面外科术前常规CTs检查不能预测多数和术中出血相关的临床事件的发生,但对术前已有凝血功能异常病史和症状、体征的患者,CTs的结果能反映凝血功能异常的程度,对预测可能出现的出血性并发症有一定的     

Study of coagulation factor activities in apheresed thawed fresh frozen plasma at 1-6 degrees C for five days

The concern for the loss of activities of coagulation factors in thawed fresh frozen plasma kept at 1-6 degrees C for long periods has prevented transfusion services from using thawed plasma beyond 24 hours of storage. There is no mention of the method of collection of the plasma and/or the study of the bacterial growth in the studies reported in the literature. The present project was undertaken to investigate coagulation factor activities and bacterial growth in apheresed fresh plasma. Twenty apheresed plasma units from different blood groups were used. After the 24-hour expiration time of the thawed plasma kept at 1-6 degrees C, aliquots were taken at day 1, day 3, and day 5 of expiration time and were immediately frozen at -70 degrees C. Samples were assayed for activities of coagulation factors II, V, VII, VIII, X, XI, and fibrinogen (Fib). Our study reveals no statistically significant change in activities of coagulation factors II, VII, X, XI, and fibrinogen from day 1 to day 5 storage of plasma at 1-6 degrees C; however, there is a mean decrease of 8.8 and 14.3% in activities of factors V and VIII, respectively. All culture samples taken on day 5 storage were negative at 7 days. In conclusion, our results do not show a significant change in the activity of most coagulation factors in the thawed apheresis plasma stored at 1-6 degrees C over a 5-day period. Hence, it is feasible to transfuse the plasma beyond the 24-hour period without compromising the clinical outcome of patients with coagulopathy.     

Measurement of blood and plasma coagulation time using free oscillating rheometry

An assay based on free oscillating rheometry to measure the activity of coagulation factors is described. The method can be used in blood and plasma and is particularly suitable for screening and monitoring coagulation disturbances in point-of-care testing (POCT) in environments where quick analysis with minimal preanalytical work is needed. In this study the endpoint as clotting onset time (COT) is determined by a deviation from initial viscoelastic properties of an oscillating sample. The model system entails the clotting of citrated blood or plasma clotting by repletion of Ca2+. COT was shown to give a dose-dependent response to added thrombin and to be resistant to high concentrations of corn trypsin inhibitor, indicating measurement of the tissue-factor-dependent pathway of coagulation activation. COT in recalcified blood and plasma covariated with prothrombin time (PT) according to Owren, and activated partial thromboplastin time (aPTT). The technique and instrument used proved to be quick and easy to handle, and suitable for POCT as well as for examinations in the laboratory.     

大量输血时血浆与红细胞的比例探讨

本研究通过回顾性分析大量输血对患者凝血功能的影响,探讨新鲜冰冻血浆与红细胞的合适比例。2011年1月-2013年1月,因各种原因需要大量输血的患者151例,按照凝血功能正常与否,分为凝血功能正常组（138例）和凝血功能异常组（13例）,以新鲜冰冻血浆与红细胞之比为1：1作为分界,每组均分为高血浆（2：1）、中血浆（1：1）、低血浆（〈1：1）3个比例组。输血前和大量输血后24h检测凝血功能。结果表明,大量输血24h后,凝血功能正常组的低血浆组凝血酶原时间（PT）、活化部分凝血活酶时间（APTT）、凝血酶时间（TT）明显延长,纤维蛋白原（FIB）含量明显降低（均P〈0．05）;高、中血浆组凝血功能无明显变化（P〉0．05）;凝血功能异常组的中血浆和低血浆组在大量输血后凝血酶原时间、活化部分凝血活酶时间、凝血酶时间,纤维蛋白原仍在异常水平（P〉0．05）,高比例组的凝血功能明显改善（P〈0．05）。结论：大量输血时,血浆与红细胞比例应根据患者凝血功能进行调整,凝血功能异常的患者建议新鲜冰冻血浆与红细胞的比例为2：1,以改善患者的凝血功能,减少不良事件的发生;凝血功能正常的患者建议冰冻血浆与红细胞的比例为1：1,减少稀释性凝血功能障碍和高血容量的发生。     

Measurement of blood and plasma coagulation time using free oscillating rheometry

An assay based on free oscillating rheometry to measure the activity of coagulation factors is described. The method can be used in blood and plasma and is particularly suitable for screening and monitoring coagulation disturbances in point-of-care testing (POCT) in environments where quick analysis with minimal preanalytical work is needed. In this study the endpoint as clotting onset time (COT) is determined by a deviation from initial viscoelastic properties of an oscillating sample. The model system entails the clotting of citrated blood or plasma clotting by repletion of Ca 2+ . COT was shown to give a dose-dependent response to added thrombin and to be resistant to high concentrations of corn trypsin inhibitor, indicating measurement of the tissue-factor-dependent pathway of coagulation activation. COT in recalcified blood and plasma covariated with prothrombin time (PT) according to Owren, and activated partial thromboplastin time (aPTT). The technique and instrument used proved to be quick and easy to handle, and suitable for POCT as well as for examinations in the laboratory.     

使用新鲜血浆建立凝血酶原时间的标准曲线

目的使用新鲜血浆建立血凝分析仪测定凝血酶原时间（PT）理想的标准曲线,以提高不同血凝分析仪测定结果的可比性。方法以校正合格的ACL-Advance全自动血凝分析仪测定的PT结果为定值,选择PT定值分别为10．5、11．0、11．5、12．0、12．5、13．0、13．5、14．0、15．0S的9个不同数值的新鲜血浆,分别对Sysmex CA-50半自动血凝分析仪建立标准曲线;然后,随机选择ACL-Advance全自动血凝分析仪已检测的PT高、中、低值患者的新鲜血浆30份,在建立标准曲线后的Sysmex CA-50半自动血凝分析仪上进行检测,并比较ACL-Advance与Sysmex CA-50血凝分析仪上测定PT（INR）结果的差异。结果使用ACL-Advance全自动血凝分析仪测定的PT结果为11．0、11．5、12．0、12．5、13．0s的新鲜血浆,建立Sysmex CA-50半自动血凝分析仪测定凝血酶原时间的标准曲线,两台仪器所检测的30份新鲜血浆的PT（INR）结果差异无统计学意义。结论用全自动血凝分析仪测定的PT为11．0～13．0s的新鲜血浆,建立半自动血凝分析仪测定凝血酶原时间的最佳标准曲线,可以明显提高不同血凝分析仪测定结果的可比性。     

Photochemical treatment of plasma with amotosalen and UVA light: process validation in three European blood centers

BACKGROUND: A photochemical treatment (PCT) process has been developed to inactivate pathogens and white blood cells (WBCs) in therapeutic plasma. Process validation studies were performed in three European blood centers under routine operating conditions. STUDY DESIGN AND METHODS: Each center prepared 30 apheresis and 30 to 36 whole blood-derived plasma units for PCT. Each whole blood-derived plasma unit contained a mixture of two to three matched donations. After removal of pretreatment control samples (control fresh-frozen plasma [C-FFP]), 546 to 635 mL of plasma was treated with 15 mL of 6 mmol per L amotosalen, 3 J per cm(2) UVA treatment, and removal of residual amotosalen with a compound adsorption device. After processing, plasma samples (PCT-FFP) were withdrawn, frozen at -60 degrees C within 8 hours of collection, and assayed for coagulation factors and residual amotosalen. RESULTS: A total of 186 units of plasma were processed. The mean prothrombin time (12.2 +/- 0.6 sec) and activated partial thromboplastin time (32.1 +/- 3.2 sec) of PCT-FFP were slightly prolonged compared to C-FFP. Fibrinogen and Factor (F)VIII were most sensitive to PCT (26% mean reduction). PCT-FFP, however, retained sufficient levels of fibrinogen (217 +/- 43 mg/dL) and FVIII (97 +/- 29 IU/dL) for therapeutic plasma. Mean levels of FII, FV, FVII, F IX, FX, FXI, and FXIII in PCT-FFP were comparable to C-FFP (81%-97% retention of activity). Antithrombotic proteins were not significantly affected by PCT with retention ranging between 83 and 97 percent. Mean residual amotosalen levels were 0.6 +/- 0.1 micromol per L. CONCLUSION: Process validation studies in three European centers demonstrated retention of coagulation factors in PCT-FFP within the required European and respective national standards for therapeutic plasma.     

存放时间和温度对凝血功能指标检测结果的影响

目的 探讨标本放置时间和温度对凝血酶原时间(PT)、活化部分凝血活酶时间(APTT)、凝血酶时间(TT)、纤维蛋白原(FⅠB)等凝血指标检测结果的影响.方法 选取抗凝静脉血标本50例,血浆用于立即测定凝血4项指标和在室温放置2 h、4 h、6 h和24 h后测定凝血4项指标;并分别于室温、-4 ℃和-20 ℃下保存24 h后测定凝血4项指标.抗凝血室温放置2 h,离心分离血浆后即刻进行凝血指标检测.采用血液凝固仪测定研究指标,采用随机单位组设计资料方差分析比较各组间差异.结果 与留取即刻检测相比,血浆放置2 h APTT、PT、TT和FⅠB等各种指标水平均无明显差异;血浆标本放置4 h开始,APTT、PT和TT等指标出现明显延长,且变化程度随放置时间延长加重,FⅠB水平无明显改变;抗凝血标本放置2 h留取的血浆标本各指标均有明显变化.-4 ℃下血浆标本保存24 h APTT、PT、TT和FⅠB等指标测定结果未出现明显改变,-20 ℃下24 h APTT出现明显延长.结论 对于凝血功能4项指标的测定,采集标本后应及时送检和尽快分离血浆.常温下血浆标本应在2 h内完成测定;-4 ℃下血浆标本保存24 h PT、APTT、TT和FⅠB等指标测定结果未受影响,低温保存应注意避免标本冻融过程.     

CA-1500血液凝固分析仪对脂血与溶血抗干扰分析

目的探讨CA-1500血液凝固分析仪对血浆标本中血红蛋白和乳脂蛋白影响的抗干扰能力。方法采用混合血浆40名正常体检人员（排除服抗凝药者）,取全血1．8ml。加入109mmoL／L的枸橼酸钠抗凝剂O．20m1混合,采血后,在1h内完成血浆的分离;以1500—2000r／min离心10min立即上机测定。结果用被检血浆重复测定凝血酶原时间（PT）、活化部分凝血活酶时间（APTT）各10次,PT（11．84±0．16）s,变异系数（CV）0．5％;APTT（31．37±0．13）s,CV0．81％。结论CA-1500血液凝固分析仪在-定浓度范围内,具有良好的抗干扰能力,不但给临床医生对病人的治疗、判断及预后提供了有效的血液凝固指标,而且为设定新的止血和血栓指标奠定了基础。     

Preservation of antithrombin III activity in stored whole blood

Antithrombin III (AT-III) is the major inhibitor of thrombin, Factor Xa, and other coagulation enzymes. Congenital and acquired deficiencies of AT-III are thought to contribute to thrombosis and disseminated intravascular coagulation. Because a recent report suggested reduced AT III in stored blood, we evaluated blood bank storage effects. Serial samples were taken from 6 units of whole blood drawn into citrate-phosphate-dextrose-adenine over 42 days, and assays for AT-III functional activity were performed on the same day. The values (mean +/- SD) were as follows: day 0,91.8 +/- 10.7 percent; day 2, 101.9 +/- 10.7 percent; day 8, 107.3 +/- 7.4 percent; day 15, 118.9 +/- 11.1 percent; day 22, 105.4 +/- 9.8 percent; day 35, 93.4 +/- 8.8 percent; and day 42, 97.4 +/- 7.5 percent. The rise from day 0 to day 15 was significant but presumably secondary to interassay variation because analysis of frozen aliquots showed no significant change when all samples from each unit were assayed in one batch. Immunoassay of AT-III also showed no change with storage. The results indicate AT-III retains functional activity in whole blood stored at 2 to 6 degrees C for 42 days, and AT-III replacement does not require fresh blood or fresh-frozen plasma. Low values may reflect individual donor differences or dilution of plasma by anticoagulant.     

全自动血凝分析仪性能验证及结果比对

目的评价新投入临床使用的1台全自动血凝分析仪(STA-R Max)性能,并与现用的全自动血凝分析仪(STA-R Evolution)进行结果比对分析。方法在2台全自动血凝分析仪上检测凝血四项[凝血酶原时间(PT)、活化部分凝血活酶时间(APTT)、纤维蛋白原(FIB)和凝血酶时间(TT)],对检测结果进行比对分析,并对新的全自动血凝分析仪精密度、线性范围和携带污染率等进行评价。结果在新的全自动血凝分析仪上重复测定正常和异常混合血浆的批间精密度,PT分别为1.12%和1.46%,APTT为0.94%和1.59%,FIB为1.83%和4.13%,TT为0.87%;批内精密度,PT为0.72%和0.44%,APTT为0.39%和0.65%,FIB为1.37%和1.75%,TT为0.87%;携带污染率为0.17%;FIB的实际检测值与理论值的线性回归方程为Y=0.9929 X-0.0937(P>0.05),截距与0之间比较差异无统计学意义(P>0.05),直线斜率与1之间比较差异无统计学意义(P>0.05);2台仪器相关性分析:PT、APTT、FIB和TT的测定结果r值分别为0.9896、0.9840、0.9951和0.9619,相对偏差分别为1.91%、2.73%、0.29%、1.51%,小于美国临床实验室改进修正法规(CLIA′88)规定的允许误差。2台全自动血凝分析仪凝血四项检测结果比较,差异均无统计学意义(P>0.05)。结论新投入使用的全自动血凝分析仪具有良好的重复性、稳定性、线性和携带污染率,2台全自动血凝分析仪结果一致,有良好的相关性,均可用于临床标本检测。