目的：对国内光学法血小板聚集功能检测现状进行调查，并进一步对标本血细胞比容对标本处理的影响、高血小板浓度的调整方式和系统间结果可比性方案进行研究并探讨用于光学法血小板聚集功能检测参考物质的研制方法，从而推进光学法血小板聚集功能检测的规范化和一致化。

针对性能验证中发现的不足，对抗凝蛋白S（PS）的日间精密度、凝血因子Ⅷ活性检测（FⅧ:C）和凝血因子Ⅸ活性检测（FⅨ:C）定量限（LoQ）进行研究，并为确定行业标准中D-二聚体的临界值是否在临床实验室有适用性，为临床实验室改善相关项目性能验证结果和完善性能验证方案提供参考。

方法：设计并发放调查问卷，了解国内血小板聚集功能检测现状，包括实验室一般信息、检测系统、检验前中后流程和质量控制等信息。参考WS/T 406《临床血液学检验常规项目分析质量要求》，使用Sysmex CN-6000进行血小板聚集功能检测重复性验证，使用高值和低值聚集率标本，以二磷酸腺苷（ADP）和胶原诱导剂连续检测10次，计算重复性以变异系数（CV）和标准差（s）表示，以满足产品说明书要求为验证通过（高值标本：CV≤20%，低值标本：s≤9%）。参考产品说明书，使用3个批号ADP和胶原诱导剂，各批号检测相同标本5次，计算15个数据的CV来表示试剂批号间差异，以满足产品说明书要求为验证通过（CV≤20%）。收集临床枸橼酸钠抗凝全血标本，参考美国临床和实验室标准协会（CLSI） H58文件、国际血栓与止血学会（ISTH）指南和法国血栓与止血学会（SFTH）指南制备富血小板血浆（PRP）与乏血小板血浆（PPP）。使用Sysmex XN-350血细胞分析仪分析同一患者血细胞比容（Hct）、全血血小板浓度和枸橼酸钠抗凝全血制备PRP的血小板浓度相关性。使用生理盐水（PS）/自体PPP调整PRP高血小板浓度标本至（250±50）和（150±50）×10⁹/L，记录血小板浓度和MA%结果进行统计分析。使用CHRONOLOG Model 700血小板聚集分析仪和CN-6000全自动凝血分析仪，分别以胶原和不同浓度ADP诱导剂，检测血小板最大聚集率（MA%），分析系统间检测结果的可比性。使用枸橼酸盐-磷酸盐-葡萄糖-腺嘌呤（CPDA）抗凝全血分离出PRP和PPP，添加保护剂Ⅱ制备用于光学法血小板聚集功能检测参考物质，参考SFTH指南和ISO GUIDE:35-2017评价稳定性。

使用CN-6000及配套试剂，使用低值和高值质控品作为研究样品。参考美国临床和实验室标准协会（CLSI）EP15-A3文件，使用3种试剂准备方式（说明书要求方式、仪器操作手册方式和改进方式）进行PS活性检测日间精密度验证研究。说明书要求方式即完全参照产品说明书开展；仪器操作手册方式在其基础上将所需试剂于仪器中静置30分钟后用于检测；改进方式在产品说明书基础上对所需试剂进行混合分装。研究同一批号试剂检测同一样品的瓶间差异，以国家卫生健康委临床检验中心（NCCL）室间质量评价（EQA）可接受范围作为评价标准。参考CLSI EP25文件，验证产品说明书方式与改进方式复溶试剂的机载稳定性。参考CLSI EP17-A2文件、WS/T 514《临床检验方法检出能力的确立和验证》和国际血液学标准化委员会（ICSH）指南，进行FⅧ:C和FⅨ:C检测LoQ验证。研究结果均以满足产品说明书要求为验证通过。参考欧洲临床化学和检验医学联合会（EFLM）生物学变异数据库数据，计算最佳水平允许总误差（TE_opt）作为D-二聚体临界值附近的浓度范围，收集临床剩余血浆并混合作为精密度验证样品。参考CLSI EP15-A3文件，使用CN-6000和STA-R Evolution进行D-二聚体临界值附近精密度验证研究。

结果：共收回有效问卷104份，实验室中使用自动化仪器共76家，占比73.1%，试剂配套比例为96.59%；标本数量＜500例/月共87家，占83.7%；申请项目的科室以神经内科、心内科和妇产科为主，申请目的以监测抗血小板药物疗效为主；68家实验室使用19～21G针头，超过80%的实验室在室温条件下直立手持运输标本；超过40%的实验室使用170g，10分钟制备PRP，超过90%的实验室使用＞1500g，10分钟制备PPP，对PRP进行血小板计数的有58家；知晓本实验室诱导剂终浓度的实验室占比＞90%，仅2家实验室使用多个浓度；超过90%的实验室报告MA%，仅20%的实验室进行室内质控，仅4家实验室参与国际室间质评计划。光学法血小板聚集功能检测ADP和胶原诱导剂的重复性（ADP：CV=3.5%，s=0.97%；胶原：CV=4.1%，s=0.50%）和试剂批号间差异（ADP：CV=6.8%；胶原：CV=6.7%）均符合产品说明书要求。全血Hct与PRP中血小板的富集倍数呈现增大趋势，Hct≥40组的PRP中血小板富集倍数显著高于Hct＜35组。高血小板浓度（>600×10⁹/L）标本经自体PPP调整后，血小板浓度显著高于生理盐水组（P<0.0001）。自体PPP调整后的MA%均被抑制（P<0.05），其中胶原诱导MA%抑制率低于ADP诱导的MA%；PS调整至（250±50）×10⁹/L组的MA%最高。检测系统间胶原诱导的MA%可比性良好（符合率=90.7%），ADP诱导结果差异显著（CN-6000使用2μmol/L ADP时符合率=42.5%，使用5μmol/L ADP时符合率=84.1%）。使用CPDA搭配保护剂Ⅱ制备的中、低聚集率水平光学法血小板聚集功能检测参考物质，可在七天内保持ADP和胶原诱导MA%稳定。

参照产品说明书与仪器操作手册进行PS活性检测精密度验证，日间精密度（CV_日间：12.9%～21.6%）均超出产品说明书要求（高值水平的CV_批内，CV_日间均＜10%，低值水平的CV_批内，CV_日间均＜20%）。应用改进方式的日间精密度验证结果（CV_日间：2.9%和4.5%），符合产品说明书要求。试剂瓶间差异超出EQA可接受范围。改进方式纠正了试剂机载稳定性验证结果（相对偏差：-4.24%～9.97%）符合产品说明书（高值水平＜10%，低值水平＜20%）。FⅧ:C和FⅨ:C检测的LoQ验证通过范围均符合厂商声明（FⅧ:C：0.75%～1.46%，0.74%～1.40%；FⅨ:C：0.71%～1.27%，0.70%～1.32%）。Sysmex CN-6000和STAGO STA-R Evolution检测系统的D-二聚体临界值附近浓度日间精密度（Sysmex：CV_日间=3.2%；STAGO：CV_日间=3.8%）均满足ICSH指南和WS/T 477-2015《D-二聚体定量检测》的要求（CV＜7.5%）。

结论：国内光学法血小板聚集功能检测的标本数量偏少，但是临床应用广泛。各实验室在光学法血小板聚集功能检测的检验前部分规范化程度较高，在检验中部分存在规范化较差，此外需要进一步研究室内质量控制和室间质量评价的开展方式，从而推进规范化和一致化进程。通过参考全血血小板浓度和Hct，可以预测PRP的血小板浓度，并对高血小板浓度的PRP推荐使用PS进行浓度调整。使用工作浓度和诱导效能相接近的诱导剂可以有效改善系统间检测结果的可比性。提供了一种光学法血小板聚集功能检测参考物质的研制方法，并初步研制出在七天内保持ADP和胶原诱导MA%稳定的光学法血小板聚集功能检测参考物质，有利于推进光学法血小板功能检测的规范化和一致化。

本研究提供了改善PS活性检测日间精密度的改进方式，并提供了FⅧ:C和FⅨ:C检测的LoQ验证方案，并提供了一种D-二聚体临界值附近浓度可接受范围的计算方式，为D-二聚体定量检测行业标准提供了临界值附近浓度日间精密度验证数据，以供临床实验室参考。

Objective: To investigate the current status of light transmission aggregation function testing in China, further investigate and develop concordance between two instruments, investigate the influence of specimen hematocrit (Hct) on platelet concentrations of platelet rich plasma (PRP), the adjustment protocols for high platelet concentrations, and explore the development method of reference material for light transmission aggregation function testing. To promote the standardization and concordance of platelet aggregation function testing.

To address problems in the verification of anticoagulant protein S (PS), coagulation factor VIII activity (FⅧ:C) and coagulation factor VIII activity (FⅨ:C) assays, as well as D-dimer performance verification, to explore improvement for the performance verification of the inter-day precision of the PS activity assay, the verification protocol of the limit of quantification (LoQ) of the FⅧ:C and FⅧ:C assays, as well as to determine whether the cut-off value of D-dimer in industry standards is applicable in clinical laboratories, and to provide a reference for clinical laboratories.

Methods: A questionnaire was designed and distributed to assess the current status of light transmission aggregation function testing in domestic laboratories, including general laboratory information, measurement systems, pre-analytical/analytical/post-analytical processes, and quality control practices. Repeatability verification for light transmission aggregation testing was conducted using the Sysmex CN-6000 analyzer in accordance with WS/T 406 “Analytical quality specifications for routine tests in clinical hematology”. High and low platelet aggregation rate samples were tested 10 times consecutively with adenosine diphosphate (ADP) and collagen as agonists. Repeatability was expressed as coefficient of variation (CV) and standard deviation (s), with verification criteria based on manufacturer specifications (high-level samples: CV ≤20%; low-level samples: s ≤9%). Reagent batches variability was evaluated using three batches of ADP and collagen agonists, with five replicates per batch. The CV of 15 measurements was calculated, and compliance with manufacturer requirements (CV ≤20%) was assessed. Citrated whole blood samples were collected,  PRP and platelet-poor plasma (PPP) were prepared following guidelines from the Clinical and Laboratory Standards Institute (CLSI) H58, International Society on Thrombosis and Haemostasis (ISTH), and Société Française de Thrombose et d’Hémostase (SFTH). The correlation between hematocrit (Hct), whole blood platelet count, and PRP platelet concentration was analyzed using the Sysmex XN-350 haematology analyser. PRP samples with high platelet concentrations (>600 ×10⁹/L) were adjusted to (250±50) ×10⁹/L and (150±50) ×10⁹/L using physiological saline (PS) or autologous PPP. Platelet counts and maximum aggregation (MA%) results were statistically analyzed. Concordance between the CHRONOLOG Model 700 platelet aggregometer and CN-6000 coagulation analyzer was evaluated for collagen- and ADP-induced MA%. Platelet aggregation testing samples with low and medium MA% were prepared using CPDA-anticoagulated whole blood supplemented with preservative agent Ⅱ. Stability was assessed according to SFTH guidelines and ISO GUIDE 35:2017. 

For PS activity inter-day precision verification, three reagent preparation methods (specification-required method, instrument's manual method and improved method) were evaluated using CLSI EP15-A3. Reagent batches variability and on-board stability were assessed using NCCL external quality assessment (EQA) criteria and CLSI EP25 respectively. LoQ verification for FⅧ:C and FⅨ:C was performed using CLSI EP17-A2, WS/T 514 “Establishment and verification of detection capability for clinical laboratory measurement procedures”, and the International Committee for Standardization in Hematology (ICSH) guidelines. D-dimer precision near cut-off value was verificated using Sysmex CN-6000 and STAGO STA-R Evolution systems, with optimal allowable total error (TE_opt) calculated based on EFLM biological variation (BV) data. 

Results: Among 104 valid questionnaires, 76 laboratories (73.1%) used automated analyzers, with 96.59% employing reagent-instrument matched systems. Most laboratories (83.7%) processed <500 samples/month. The primary clinical departments requesting testing included neurology, cardiology, and obstetrics/gynecology, mainly for antiplatelet therapy monitoring. Needle sizes (19–21G), upright room-temperature transport (＞80%), PRP preparation (170g, 10 min; ＞40%), and PPP preparation (＞1500g, 10 min; ＞90%) were common. Only 58 laboratories performed PRP platelet counts, and ＞90% reported MA% without internal quality control (20%) or international EQA participation (4 laboratories). ADP- and collagen-induced aggregation repeatability (ADP: CV=3.5%, s=0.97%; collagen: CV=4.1%, s =0.50%) and variation of 3 batches (ADP: CV=6.8%; collagen: CV=6.7%) met manufacturer criteria. PRP platelet enrichment correlated positively with Hct, with significantly higher enrichment in Hct ≥40% vs. Hct <35% groups (P<0.05). Autologous PPP-adjusted PRP exhibited higher platelet counts (P<0.0001) and suppressed MA% (P<0.05), with collagen-induced MA% inhibition lower than ADP-induced. PS-adjusted PRP ([250±50]×10⁹/L) showed the highest MA%. Inter-system agreement was strong for collagen-induced MA% (90.7%) but variable for ADP-induced results (42.5% at 2 μmol/L vs. 84.1% at 5 μmol/L). preservative agent Ⅱ-stabilized reference material maintained stable ADP- and collagen-induced MA% for seven days. 

PS activity verification using manufacturer protocols yielded unsatisfactory inter-day precision (CV: 12.9%–21.6%). Improved reagent preparation reduced inter-day CV to 2.9%–4.5%, meeting specifications. Variation of the bottle variation of the same batch of reagents exceeded EQA limits. Improved protocols ensured on-board stability compliance (relative bias: -4.24%–9.97%). LoQ ranges for FⅧ:C (0.74%–1.46%) and FⅨ:C (0.70%–1.32%) aligned with manufacturer claims. D-dimer inter-day precision (Sysmex: CV=3.2%; STAGO: CV=3.8%) satisfied ICSH and WS/T 477-2015 requirements (CV<7.5%). 

Conclusion: Platelet aggregation testing in China, though limited in sample volume, is widely applied clinically. Pre-analytical standardization is relatively advanced, while analytical variability remains significant. Internal quality control and EQA require further standardization. PRP platelet concentration can be estimated from whole blood counts and Hct. PS is recommended for PRP adjustment. Harmonizing agonist concentrations improves inter-system consistency. A method for developing light transmission aggregation reference material was established. The reference material of light transmission aggregation stable for seven days, and support standardization efforts.

This study provides improved ways to improve the inter-day precision of the PS activity assay, and provides a LoQ verification protocol for the FⅧ:C and FⅨ:C assays, and provides a calculation of the acceptable range of concentrations near the D-dimer cut-off value, which provides data for the industry standard for quantitative D-dimer assays for the inter-day precision of the concentrations near the cut-off value for the reference of clinical laboratories.