Performance Evaluation And Clinical Utility Of An Enzymatic Method For The Measurement Of RLP-C

Objective:Remnant lipoprotein cholesterol(RLP-C),also known currently as the products of triglyceride-rich lipoprotein particles remnants,are closely associated with atherosclerotic cardiovascular disease(ASCVD).The aim of this study is to verify the analytical performance of RLP-C-enzymatic quantitative measurement system and to explore the clinical utility of screening ASCVD and predicting coronary artery stenosis by RLP-C.Methods:1.Based on the approved guidelines of CLSI EP15-A2,CLSI EP6-A,CLSI C28-A2 and other reference criteria of performance evaluation,we verified the precision,linearity,accuracy and reference interval of RLP-C enzymatic quantitative measurement system.2.Testing RLP-C levels in serum among ASCVD patients and healthy population by RLP-C enzymatic quantitative measurement system to further study the cut-off value of RLP-C used to diagnose ASCVD by ROC curves.3.Analyzing the correlation between RLP-C levels and coronary stenosis degree with Gensini score,to find independent risk factors of coronary stenosis and establish logistic regression model based on these factors.4.Evaluation of the consistency between enzymatic quantitative measurement and Friedewald equation estimation was performed respectively by the Bland-Altman plots and the ICC.5.Correlation coefficient matrix diagram was plotted to analyze the relationship of the components of lipoprotein cholesterol.The composition characteristics of each lipoprotein cholesterol component at different TG levels was covered in a more depth fashion to investigate the bias between the determined RLP-C by enzymatic method and the estimated value of Friedewald equation.Results:1.RLP-C enzymatic quantitative measurement system has good precision,linearity and accuracy.Repeatability imprecision,middle imprecision and total imprecision CV of RLP-C in the samples with a lower RLP-C levels were 4.90%,3.70%and 5.40%respectively.These values were 1.30%,1.30%and 1.70%in the samples with higher RLP-C levels.The results of linearity indicated RLP-C are linear in the related concentration range(3.90～53.40mg/dL),with a linear regression equation:Y=0.794+0.994X(R2=0.998).The data of trueness evaluation showed the bias of results were 7.70%and 9.40%,which below 10%given by the reference laboratory.The reference interval verification pointed out that only one individual’s RLP-C level was above of 12.0mg/dL in 20 reference observations so that the 95%reference interval given by the reference laboratory can be received.2.The serum RLP-C level of 671 ASCVD patients was higher than that of 325 healthy individuals with concentrations of 10.3 mg/dL[IQR(6.4,15.5)]and 9.2 mg/dL(6.8,11.9)(P<0.001)respectively.ASCVD patients were further divided into peripheral atherosclerosis group and coronary heart disease(CHD)group,but there was no significant difference in serum RLP-C between the two group[10.7 mg/dL(6.6,15.4)vs.9.9 mg/dL(5.9,15.9),P=0.416].The cut off value of screening ASCVD and CHD were 11.75 mg/dL and 12.70 mg/dL respectively with the poor sensitivity and specificity.3.There was a positive correlation between RLP-C levels and Gensini score of patients with CHD(r=0.75,P<0.001).Multivariate logistic regression analysis showed that serum RLP-C,smoking,hypertension,diabetes,and monocyte/HDL-C ratio were risk factors for the severe CHD.Based on these risk factors,we constructed a predictive model to discriminate severe coronary stenosis,of which the sensitivity and specificity were 87.5%and 81.0%respectively(logit P=-8.07+1.21×smoking history+1.11×hypertension history+1.26×diabetes history+0.29×RLP-C+1.85×monocytes/HDL-C).4.The Bland-Altman plot showed that 7.0%(70/996)points was located outside the 95%limits of agreement(95%LoA),and the Friedewald equation estimation was 0.2 mmol/L(95%CI,0.18～0.23)higher per 1.0mmol/L than enzymatic method.ICC method showed that ICC=0.551[(95%CI,0.192～0.732),P<0.001],which indicated a poor consistency of two method.RLP-C estimated by the Friedewald equation is significantly correlated with the RLP-Cm(r=0.4,P<0.001).However,as the RLPCe increase gradually,the difference between RLP-Ce and RLP-Cm is increasing(P<0.001),with an average positive bias of 0.2 mmol/L.5.The lipoprotein cholesterol correlation matrix showed a significant negative correlation between RLP-C and HDL-C(rs=-0.2,P<0.001)and a significant positive correlation with LDL-C,TC,TG,non-HDL-C(P<0.001),among which the correlation with TG is strongest.The further study found that with the increase of TG concentration,RLP-Ce and RLP-Cm both rise significantly and the RLP-Cm/RLP-Ce ratio decreased gradually.Conclusion:A good analytical performance of RLP-C enzymatic quantitative measurement system was found which could meet clinical needs.RLP-C are closely related to the pathogenesis of ASCVD and severity of CHD.Our finding emphasize the significance to standardize definition and build measurements of RLP-C.