第一部分 纤维蛋白原和抗凝蛋白AT、PC检测参考物质互通性评价

及相关问题研究

目的：

评价纤维蛋白原（Fib）、抗凝血酶（AT）、蛋白C（PC）检测世界卫生组织（WHO）国际标准品、国际血栓与止血协会科学标准化委员会（SSC/ISTH）凝血标准品（SSC LOT4）及自制参考物质在常用检测系统间的互通性，为实验室合理选择互通性评价方法及可靠的溯源标准提供参考。

方法： 

1.样本冻融影响及冻存样本复融后室温放置时间研究：收集15例临床样本，分别于样本冻存前、复融后即刻、室温放置2h、4h时检测Fib、AT、PC。计算样本冻存前后检测结果偏差范围，评价冻融对检测的影响；计算样本室温放置2h时、4h时与复融即刻时检测结果的差异，评价冻存样本复融后室温放置时间。

2.检测系统的可比性评价及改进：检索生物学变异（BV）文献，依据欧洲临床化学和检验医学联合会（EFLM）发布的BV数据核查表（BIVAC）对文献进行筛选和评价，计算基于可靠BV数据推导的不同水平的偏倚（Bias）并总结不同质评机构室间质量评价（EQA）允许范围，作为评价标准制定的参考依据。在不同检测系统上同时检测临床样本，检测系统两两比较，进行可比性评价。评价标准优先使用基于可靠BV数据推导的评价标准，若临床样本检测结果不能满足基于BV推导的标准，则使用EQA允许范围的一半作为评价标准，根据各检测项目的数据分布特征分别设置适当的符合率要求。若各检测系统配套商品校准品校准条件下，样本检测结果的可比性无法满足评价标准要求，则使用WHO国际标准品为检测系统校准，进行可比性改进。

3.参考物质的互通性评价：检测系统的可比性满足评价标准要求的基础上，将待评价参考物质随机穿插于临床样本间，在不同检测系统上同时检测。参考美国临床实验室标准化协会（CLSI）EP14-A3文件和国际临床化学和实验室医学联合会（IFCC）互通性评价工作组的推荐方法。分别采用Deming回归法及偏差差值评估法评价Fib、AT、PC检测WHO国际标准品、SSC LOT4、自制参考物质的互通性。

结果：

1.样本冻融影响及冻存样本复融后室温放置时间研究：15例样本冻融前后，Fib检测相对偏差范围是-4.9%~0，AT活性检测相对偏差范围-4.7%~-1.2%，PC活性检测偏差范围是-3.5%~0.4%，样本冻融前后检测差异较小，认为冻融对检测结果无影响；冻存样本复融后室温放置4h时，与复融后即刻检测结果相比，Fib的偏差范围为0~4.6%，AT的偏差分布范围是2.3%~6.0%，PC的偏差分布范围是2.7%~7.5%，冻存样本复融后室温放置4h变化不大，故Fib、AT、PC检测冻存样本复融后至少可稳定4h。

2.检测系统的可比性评价和改进及评价标准的确定：Fib和AT检测，厂家配套校准方式下检测临床样本，基于BV推导的评价标准及EQA允许范围的一半均无法满足。实施可比性改进后，系统间样本检测结果满足BV推导的评价标准的比例仍不足80%，使用RCPA的EQA允许范围的一半作为评价标准，Fib样本符合率80%以上，AT可达到85%以上。PC活性检测，各检测系统在厂家各自配套的校准方式下检测临床样本，系统间样本检测结果的偏差可满足基于BV研究的最低偏倚的要求，满足评价标准的样本比例可达到90%及以上。

3.参考物质的互通性评价：Deming回归法与偏差差值评估法评价参考物质互通性，结论存在差异。Deming回归法互通性评价的允许范围受检测系统可比性和精密度影响，偏差差值评估法考虑了临床需求和参考物质的预期用途，是一种较为合理的互通性评价方法。偏差差值评估法参考物质互通性评价结果显示，Fib检测，WHO 09/264在Stago和Sysmex两个检测系统间，Sysmex和IL两个检测系统间具有互通性，在Stago和IL两个检测系统间的互通性难以确定（WHO 09/264的校准效果表明，其在Stago和IL两个检测系统间具有互通性），SSC LOT4在Stago和Sysmex两个检测系统间具有互通性，在Stago和IL两个检测系统间、Sysmex和IL两个检测系统间的互通性难以确定，自制参考物质Fib-RM01、Fib-RM02在三个检测系统间具有互通性，Fib-RM03在Stago和Sysmex两个检测系统间不具有互通性，在Stago和IL两个检测系统间、Sysmex和IL两个检测系统间的互通性难以确定；AT活性检测，WHO 08/258、SSC LOT4、自制参考物质在三个检测系统间均具有互通性；PC活性检测，WHO 02/342、SSC LOT4、自制参考物质在三个检测系统间均具有互通性。

结论：

Fib、AT、PC检测，冻融一次对样本检测影响不大，冻存样本复融后室温可稳定至少4h。参考物质的互通性评价需要在检测系统的可比性满足要求的基础上进行，不同的评价方法，互通性评价结果存在差异，推荐使用偏差差值评估法评价参考物质的互通性。Fib、AT、PC检测WHO国际标准品和正常水平自制参考物质在常用检测系统间具有互通性，可作为量值溯源的标准，用于实现标准化。

第二部分 老年患者关节置换术后低分子量肝素抗凝治疗监测初步研究

目的：  

本研究旨在研究老年患者关节置换术后低分子量肝素预防性用药抗Xa水平分布范围及D-二聚体（D-D）、纤维蛋白（原）降解产物（FDP）动态变化情况，探讨血栓、出血等不良事件发生的危险因素。

方法：

1.制定并实施研究方案：通过文献检索与学习，设置研究对象纳入和排除标准、样本采集时间、明确需要重点关注的临床信息等。通过与临床沟通交流，结合实际情况，确定具体实施方法，包括样本采集方式、不良事件的判断方法等。本研究的研究对象为全髋关节置换术、全膝关节置换术、股骨头置换术后使用依诺肝素抗凝，年龄大于60岁的患者；抗Xa活性检测样本采集时间为稳态峰值及谷值；D-D、FDP术前、术后动态监测。筛选入组人群，规范采集样本并冻存、采集临床信息，记录患者发生的血栓和出血事件。冻存的临床样本进行集中检测。

2.数据分析：根据数据分布情况，选择合适的统计方法。分别按照指标检测结果、不良事件结局分组统计，判断检测指标与不良事件发生的相关性。

结果：

目前已入组8例患者，8例患者均采集到抗Xa峰值样本，仅5例患者采集到谷值样本。抗Xa峰值水平分布范围为0.36~0.56IU/ml，谷值水平分布范围为0.10~0.13IU/ml。患者围手术期D-D、FDP呈现动态变化的趋势，术前约有一半的患者D-D、FDP高于正常水平。术后第1天D-D、FDP值最高，术后第3天降低，部分患者降低后有再次升高的趋势。

结论：

目前入组人群较少，未能得出一般性结论，后续需增加样本例数。进行研究。

part 1 study on commutability evaluation of reference materials for fibrinogen and anticoagulant proteins at and pc

ives: to evaluate the commutability of the who international standard, ssc/isth secondary coagulation standard and homemade reference materials among common measurement systems in order to give suggestions on how to determine the suitable method of commutability evaluation and reliable traceability standard.

methods:

1.study on the effect of frozen-thawed samples and the stable time on temperature: 15 fresh individual samples were collected and the fib, at and pc of each sample were measured before frozen, after thawed immediately, 2 and 4 hours after thawed at room temperature respectively. the differences between the measurement results before frozen and after thawed were calculated, as well as the differences between 2 hours, 4hours at room temperature and at once after being thawed. the effect of freeze-thawed on fib, at and pc measurement results and stable time of thawed samples were evaluated.

2.comparability evaluation and improvement of measurement systems: bv publications were searched and rated according to biological variation data critical appraisal checklist (bivac) published by european federation of clinical chemistry and laboratory medicine (eflm). different levels of bias based the reliable bv data were calculated and the allowable deviations of external quality assessment (eqa) were summarized. clinical samples were measured simultaneously on different measurement systems to evaluate the comparability. the criterion were determined based on reliable bv data. if the results of clinical samples can not meet the criterion based on bv, half of the eqa allowable deviations is used as criterion. according to the results of clinical sample, appropriate agreement rate requirements are set respectively. the who international standard was used to calibrate each system to improve the agreement if the comparability among different systems can’t be accepted.

3.commutability evaluation of reference materials: on the premise that the comparability was accepted, clinical samples and the reference materials randomly interspersed among the clinical samples were measured on different measurement systems simultaneously. measurement results were pairwise analyzed by deming regression and difference in bias approach according to the clinical and laboratory standards institute (clsi) ep14-a3 protocol and the recommendations of the international federation of clinical chemistry and laboratory medicine (ifcc) working group on commutability, respectively.

results:

1.study on the effect of frozen-thawed and the stable time on temperature: measurement results showed that the relative bias of the 15 samples before and after once frozen-thawed ranged from -4.9% to 0 for fib, -4.7% to -1.2% for at, -3.5% to 0.4 for pc. it is considered that once frozen-thawed of clinical samples have no effect on the measurement results. the relative bias between 4 hours at room temperature after thawed and immediate measurement ranged from 0 to 4.6% for fib, 2.3% to 6.0% for at, 2.7% to 7.5% for pc. it is considered that the sample were stable at least 4 hours at room temperature after thawed.

2.comparability evaluation and improvement of measurement systems: for fib and at, the comparability among the different measurement systems can not meet the criterion of bv-bias or the half of eqa criterion. after being calibrated with who international standard, the proportion of sample meeting the bv-bias_min is still less than 80%. using half of the eqa allowable range of rcpa as the evaluation criterion, the compliance rate of fib samples is more than 80%, and at can reach more than 85%. for pc, the measurement results of 90% sample between different systems can meet bv-bias when calibated with corresponding calibrators.

3.commutability of reference materials: there are differences in the results of two commutability evaluation approaches. the prediction interval of deming regression was affected by the comparability and precision of measurement systems. the difference in bias approach is more suitable because its criterion is related to the medical requirements and intended use. who 09/264 was commutable between stago and sysmex, sysmex and il, inconclusive between stago and il (the calibration effectiveness of who 09/264 show that it was commutable between stago and il). ssc lot4 was commutable between stago and sysmex, inconclusive between stago and il, sysmex and il. homemade reference materials, fib-rm01 and fib-rm02 were commutable between all systems pairs, fib-rm03 was not commutable between sysmex and stago, inconclusive between stago and il, sysmex and il for fib, who 08/258, ssc lot4, homemade reference materials were commutable between all systems pairs for at, who 02/342, ssc lot4, homemade reference materials were commutable between all systems pairs assessed by difference in bias approach for pc.

conclusions:

once frozen-thawed of clinical samples have no effect on the measurement results and the sample were stable at least 4 hours at room temperature after thawed for fib, at, pc.

after the comparability can be accepted, the commutability evaluation of reference materials can be evaluated. different commutabilty evaluation approaches may lead to different results. the difference in bias approach is recommended. who international standard and homemade reference materials in normal range were commutable among common measurement systems and can be used as traceability standard for fib, at and pc. 

part 2 preliminary study on laboratory monitoring of low-molecular-weight heparin anticoagulant therapy in elderly patients after total joint arthroplasty

ives: to investigate the distribution range of anti-xa levels and the perioperative changes of d-dimer (d-d) and fibrin/fibrinogen degradation products (fdp) in elderly patients after total joint arthroplasty with low molecular weight heparin (lmwh) prophylaxis venous thromboembolism (vte) and explore the risk factors of bleeding and thrombosis.

methods:

development and implementation the research plan:the subject inclusion and exclusion criteria, sample collection time and clinical information were determined after literature searching and learning. the sample collection methods was determined after communication with the clinicians. the ives were elderly patients over 60 years old after total keen arthroplasty, total hip arthroplasty or artificial femoral head replacement with enoxaparin prophylaxis vte. anti-xa activity sample collection and measurement time is steady state peak and trough level. the collection time of d-d、fdp were before surgery and after surgery. the enrolled patients were screened and the clinical information were collected. the samples were collected and measured and the thrombosis or bleeding events were recorded. the collected clinical samples were measured together.

data analysis:the appropriate statistical method was chose depended on the data distribution. the results were analyzed to determine the correlation between the measurement results and the thrombosis or bleeding events.

results:

8 patients have been enrolled which had been collected peak level, only 5 patients have collected trough level. the peak level of xa ranged from 0.36 iu/ml to 0.56 iu/ml, and the trough level ranged from 0.10 iu/ml to 0.13 iu/ml. not all patients can collect samples at all time point. d-d and fdp in the perioperative period showed a dynamic change. about half of patients had higher d-d and fdp than the reference range before surgery. the d-d and fdp were highest on the first day after surgery, and decreased on the third day after surgery. some patients increased again after the decrease.

conclusions: the number of sample cases is small now and no general conclusion can be drawn.