基于Mg2+-Ca2+的APTT用于检测狼疮抗凝物质的新方法的建立及应用评价

摘要

目的 建立并评价基于Mg2+/-Ca2+联合测定活化部分凝血活酶(APTT)用于检测狼疮抗凝物质(LA)的新方法.方法 本前瞻性研究共纳入244例随机患者和65例不同自身免疫性疾病患者柠檬酸三钠抗凝血浆分别用于方法建立和评价.应用Actin试剂,分别以0、2、4、8、16 mmol/L Mg2+联合25 mmol/L Ca2+溶液(Mg2+-Ca2+溶液)测定94例患者血浆APTT值,观察Mg2+对LA阴性和阳性血浆APTT的影响并确定Mg2+-Ca2+溶液试验浓度(试验溶液).采用Actin试剂分别以试验溶液和25 mmol/L Ca2+溶液测定患者和正常血浆APTT,计算两种溶液测定的APTT比值(Mg2+-Ca2+-APTT指数)和标准化Mg2+-Ca2+-APTT比值(NAR);测定混合血浆NAR并计算其CV%用于评价该方法的重复性和稳定性.随机选取150例测定凝血试验的患者血浆,以前述试验溶液及Actin试剂测定APTT,计算Mg2+-Ca2+-APTT指数并以稀释蝰蛇毒试验(dRVVT)法检测血浆标准化LA比值(NLR),比较Mg2+-Ca2+-APTT指数与NLR的关系以验证试验溶液的适用性.最后以Mg2+-Ca2+联合测定APTT法和dRVVT法分别检测65例自身免疫性疾病(26例SLE)患者NAR和NLR,以dRVVT法为标准运用ROC曲线评估2种方法检测LA的效能.结果 在0~16 mmol/L Mg2+-Ca2+溶液范围内,所有LA阴性血浆中,APTT正常组APTT从28.1 ±4.5 s升高到57.7 ±6.4 s,APTT延长组从47.2 ±8.9 s升高到97.5 ±10.3 s,ACA阳性组从27.6 ±5.1 s升高到61.2 ±7.9 s(F=34.12、38.9和28.35,P<0.01);LA阳性APTT正常和延长者APTT值在0~4 mmol/L范围随Mg2+浓度升高而缩短,在4~8 mmol/L范围则延长(F=31.55和39.5,P<0.01),8.0 mmol/L Mg2+时APTT显著高于4.0 mmol/L的APTT值(P<0.001);确定试验浓度为4.0 mmol/L.试验溶液测定NAR的批内、批间和天间CV%分别为1.39%、2.30%和3.44%.分别以<0.966和>1.034为判断标准,150例患者中141例Mg2+/Ca2+-APTT指数>1.034的血浆均NLR<1.20,9例<0.966者血浆NLR≥1.20.NAR检测LA的ROC曲线下面积(AUC)为0.913(95% CI:0.848~0.978),敏感度85%,特异度77%, cut-off值为0.877;NLR的AUC为0.892(95% CI:0.817~0.966),敏感度81%,特异度74%,cut-off值为1.13;两种方法的阳性符合率为94.4%,阴性符合率为98.5%,总符合率为98%.结论 基于Mg2+-Ca2+联合测定APTT检测LA的方法具有可行性,并可初步应用于LA检测.%Objective To establish and assess the new method of APTT assay based on the combinations of Mg2+and Ca2+for lupus anticoagulants(LA)measurements.Methods This prospective study included 309 trisodium citrate anticoagulated plasma samples from 244 random patients and 65 patients with different autoimmune diseases(AID)to establish and assess the method of LA measurement, respectively.Final concentrations of 0,2.0, 4.0, 8.0,16.0 mmol/L Mg2+were added into 25 mmol/L Ca2+solution, and Actin reagent was used to measured plasma APTT of 94 patients.The applied concentration of Mg2+-Ca2+solution was confirmed through the special and significant alteration of APTT from LA-positive and -negative plasma observed in the presence of Mg 2+(test solution).Based on Actin reagent use,the test solution and 25 mmol/L Ca2+solution were applied to measure APTT of patients and normal individuals, respectively, and the ratio of Mg2+-Ca2+-APTT to Ca2+-APTT(Mg2+-Ca2+-APTT indices)and normalized Mg2+-Ca2+-APTT indices(NAR)were calculated, respectively.Mixed plasma NAR was measured,and CV%was calculated to evaluate the repeatability and stability of Mg 2+-Ca2+-APTT method.APTT of 150 patients was measured with the test solution and Actin reagent to calculate Mg 2+-Ca2+-APTT indices, and normalized LA ratio was determined with dRVVT method.The applicability of Mg2+-Ca2+-APTT assay was assessed through comparisons of the results from the two methods.Finally, NAR and NLR of 65 patients with AID(including 26 SLE patients)were measured with Mg2+-Ca2+-APTT assay and dRVVT method, respectively, and ROC curve was also used to assess the efficacy of the two methods for LA measurements.Results In all LA-negative plasma,APTT increased from 28.1 ±4.5 s to 61.2 ±7.9 s in normal APTT group,47.2 ±8.9 s to 97.5 ±10.3 s in increased APTT group,and 27.6 ± 5.1 s to 61.2 ±7.9 s in ACA-positive group when Mg2+increased from 0 to 8 mmol/L in Mg2+-Ca2+solution(F=34.12, 38.9 and 28.35,P<0.01).Following increased Mg2+concentration, APTT shortened from 0 to 4.0 mmol/L, but simultaneously prolonged from 4.0 to 16.0 mmol/L in LA-positive plasma with prolonged or normal APTT(F=31.55 and 39.51, P<0.01), and APTT was significantly higher in 8.0 mmol/L than that in 4.0 mmol/L(P<0.001).The test concentration of Mg 2+/Ca2+solution was 4.0 mmol/L.The within, inter-day CV% of NAR was 1.39%,2.30%, and 3.44%, respectively. According to the judging criteria of <0.966 and >1.034 of Mg2+/Ca2+indices, there was 141 patients with increased indices and NLR <1.20, and 9 patients with decreased ones and NLR≥1.20 in all 150 patients.The area under ROC curve of NAR and NLR for LA detection was 0.913(95%CI:0.848-0.978) and 0.892(95%CI:0.817-0.966), respectively, and the cut-off value was 0.87 and 1.13, respectively. The sensitivity and specificity of NAR(85% and 77%)was higher than that of NLR(81% and 74%), respectively.The accordant rate of positive,negative,and total results between NAR and NLR was 94.4%, 98.5%,and 98%,respectively.Conclusion The method of APTT assay based on Mg2+combining Ca2+for LA measurements is feasible,and can be used to detect plasma LA of patients.