诊断学理论与实践

诊断学理论与实践 ›› 2026, Vol. 25 ›› Issue (02): 200-208.doi: 10.16150/j.1671-2870.2026.02.011

• 论著 • 上一篇    下一篇

轻型血友病A患者采用一期法与发色底物法检测凝血因子Ⅷ促凝活性结果差异分析及机制研究

陈光明a,b, 林莉亚a, 吴灏洋a, 武文漫a, 丁秋兰a, 王学锋a(), 戴菁a()   

  1. a 上海交通大学医学院附属瑞金医院 检验科 上海血液学研究所， 上海 200025
    b 上海交通大学医学院附属瑞金医院 医学基因组国家重点实验室 上海血液学研究所， 上海 200025
  • 收稿日期:2026-02-10 修回日期:2026-03-16 接受日期:2026-03-20 出版日期:2026-04-25 发布日期:2026-04-25
  • 通讯作者: 王学锋 E-mail：wangxuefeng6336@hotmail.com；
    戴菁 E-mail：dj40572@rjh.com.cn
  • 基金资助:
    国家自然科学基金面上项目(82170128);国家重点研发计划(2023YFC2507800)

Analysis of discrepancies between one-stage clotting assay and chromogenic substrate assay for coagulation factor Ⅷ activity and their underlying mechanisms in patients with mild hemophilia A

CHEN Guangminga,b, LIN Liyaa, WU Haoyanga, WU Wenmana, DING Qiulana, WANG Xuefenga(), DAI Jinga()   

  1. a Department of Laboratory Medicine, Shanghai Institute of Hematology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
    b State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
  • Received:2026-02-10 Revised:2026-03-16 Accepted:2026-03-20 Published:2026-04-25 Online:2026-04-25

RichHTML

2

PDF (PC)

16

摘要：

目的：一期法检测（one-stage clotting assay，OSA）测定凝血因子Ⅷ活性（FⅧ：C）是目前临床最常用的方法，但在携带特定错义突变的血友病A患者中，OSA测得的FⅧ：C水平可能高于临床出血表型所提示的水平，导致疾病严重程度被低估，甚至造成血友病的漏诊，而发色底物法检测（chromogenic substrate assay，CSA）常用于OSA的补充。本研究分析轻型血友病A（mild hemophilia A， MHA）患者的基因型及实验室表型，并探讨OSA、CSA及凝血酶生成试验（thrombin generation test，TGT）在MHA诊断中的应用价值。方法：2019年6月至2025年9月，回顾性收集我院疑诊为MHA，且OSA的FⅧ：C水平与实际出血表型不符的男性患者5例，为连续入组，患者年龄范围为24~75岁。收集所有患者临床资料，采用OSA和CSA检测患者的FⅧ：C，并比较2种方法的检测结果间的差异；采用TGT评估患者的整体凝血潜力；采用二代测序结合桑格测序确定患者的F8基因型，并采用PyMOL软件对FⅧ结构域及关键氨基酸残基进行可视化分析，探讨突变对FⅧ空间结构的影响。结果：OSA提示，5例患者（A-E）中，3例（A、C、E）患者的FⅧ：C<40% ，而2例（B、D）患者FⅧ：C分别为55.3%、63.7%。CSA结果提示，5例患者CSA的FⅧ：C水平在7.8%~25.7%之间，5例患者均诊断为MHA。OSA结果/CSA结果比值为1.3~5.5，CSA结果更符合患者的临床出血表型。5例患者中，4例进行了TGT，与正常人混合血浆相比，患者的凝血酶生成潜力、凝血酶峰值及凝血酶生成速率均降低，达峰时间延长。5例患者中，共检出4种已报道的F8错义突变，分别为p.V285M（患者A）、p.A303P（患者C）、p.S308L（患者B、D）、p.R546W（患者E）。PyMOL软件分析提示，前3个突变位于FⅧ的A1、A3与A2结构域相互作用界面，可能通过影响活化的FⅧ（FⅧa）的稳定性，造成OSA检测结果与临床出血表型不一致。p.R546W突变则位于该相互作用界面附近，对FⅧa稳定性的影响相对较小。结论：OSA测得的FⅧ：C水平可能与部分MHA患者的临床出血表型不一致，联合应用OSA和CSA有助于提高诊断准确率。TGT可用于辅助评估患者的整体凝血功能，为疾病精准诊断提供参考。

关键词: 轻型血友病A, 实验室诊断, 检测差异, 凝血酶生成试验

Abstract:

Objective The one-stage clotting assay (OSA) for measuring coagulation factor Ⅷ activity (FⅧ:C) is currently the most commonly used method in clinical practice. However, in patients with hemophilia A carrying specific missense mutations, the FⅧ:C level measured by OSA may be higher than that indicated by the clinical bleeding phenotype, leading to underestimation of disease severity or even missed diagnosis of hemophilia. The chromogenic substrate assay (CSA) is often used as a supplement to OSA. This study analyzes the genotypes and laboratory phenotypes of patients with mild hemophilia A (MHA) and explores the diagnostic value of OSA, CSA, and thrombin generation test (TGT) in MHA. Methods From June 2019 to September 2025, five male patients suspected of having MHA, whose FⅧ:C levels measured by OSA were inconsistent with their actual clinical bleeding phenotype, were retrospectively and consecutively enrolled at our center. The patients' ages ranged from 24 to 75 years. Clinical data were collected from all patients. FⅧ:C levels were measured using both OSA and CSA, and the differences between the two assays were compared. TGT was performed to evaluate the patients' global coagulation potential. The F8 genotype was determined using next-generation sequencing combined with Sanger sequencing. Structural visualization of FⅧ domains and key amino acid residues was performed using PyMOL software to explore the effects of mutations on the spatial structure of FⅧ. Results OSA indicated that among the five patients (A-E), three patients (A, C, and E) had FⅧ:C levels <40%, while the other two patients (B and D) had FⅧ:C levels of 55.3% and 63.7%, respectively. CSA results showed that the FⅧ:C levels of the five patients ranged from 7.8% to 25.7%, and all five patients were diagnosed with MHA based on CSA results. The ratio of OSA to CSA results ranged from 1.3 to 5.5, and CSA results were more consistent with the clinical bleeding phenotype in patients'. TGT was performed in four of the five patients. Compared with normal pooled plasma, the patients showed reduced endogenous thrombin potential, decreased peak thrombin, and lower thrombin generation velocity, along with prolonged time to peak. Four previously reported F8 missense mutations were identified among the five patients: p.V285M (patient A), p.A303P (patient C), p.S308L (patients B and D), and p.R546W (patient E). PyMOL structural analysis indicated that the first three mutations were located at the interaction interface of the A1, A3, and A2 domains of FⅧ, potentially affecting the stability of activated FⅧ (FⅧa) and causing inconsistency between OSA results and clinical bleeding phenotype. The p.R546W mutation was located near this interaction interface and had a relatively smaller effect on FⅧa stability. Conclusions FⅧ:C levels measured by OSA may be inconsistent with the clinical bleeding phenotype in some patients with MHA. The combined use of OSA and CSA can help improve diagnostic accuracy. TGT can be used to assist in evaluating patients' global coagulation function and provide a reference for precise diagnosis.

Key words: Mild hemophilia A, Laboratory diagnosis, Assay discrepancy, Thrombin generation test

中图分类号: