Establishment And Application Of The Reference Intervals Of The Cardiac Markers

Objective:According to the World Health Organization(WHO),cardiovascular disease(CVD)has become the leading cause of death worldwide.The 2018 National Report showed that China's CVD prevalence is in a sustained rise.The annual deaths due to CVD is higher than ones due to tumor or other diseases and ranks the first among all diseases.Cardiac markers play important laboratory roles in the prevention,early diagnosis and treatment,therapy efficacy evaluation and prognosis of CVD.Although,the classical cardiac markers,Creatine phosphokinase MB isoform(CK-MB)and myoglobin(Mb),are no longer the primary cardiac markers for ACS diagnosis,but they are still widely used in Chinese clinical laboratories,as high as 61.3% and 98% Chinese clinical laboratories respectively.High sensitivity cardiac troponin(hs-c Tn)including two subtypes of hs-c Tn T and hs-c Tn I,has been defined as the preferred cardiac marker in the Fourth universal definition of myocardial infarction published in 2018 and the non-ST segment elevation acute coronary syndrome guidelines published by European Society of Cardiology in 2015 currently.The N-terminal B-type natriuretic peptide(NT-pro-BNP)has been recommended as an important biomarker for the diagnosis,treatment,and therapy efficacy evaluation for both Acute and Chronic Heart Failure,according to the Chinese National Guidelines(2014)for Non-ST-Severe Acute Coronary Syndromes(ACS)and the European Society of Cardiology(ESC)Guidelines 2016 for the diagnosis and treatment of acute and chronic heart failure.Appropriate reference population is very important for establishing reference interval.However,up to now,there is no international consensus on how to define reference population,which is also the difference and difficulty between establishing reference interval of myocardial markers and other clinical testing items.In 2018,the recent published clinical laboratory practice recommendations for the use of c Tn in ACS: expert opinion from the academy of the American Association for Clinical Chemistry and IFCC TF-CB(we call it 2018 AACC/IFCC criteria for short)was released,but there are still deficiencies,ignoring the important cardiovascular risks factors such as hypertension,overweight and obesity,and dyslipidemia.Not match to the importance and widely usage of the cardiac markers,the reference intervals of cardiac markers currently used in different hospitals or clinical laboratories in China are obviously different,and the original source of the reference intervals are not unified.Most laboratories use the reference intervals recommended by the manufacturers' instructions,which were mostly established based on the studies done for the European and American population.Due to possible racial differences,directly applying these reference intervals on Chinese population is questionable.Establishing reference intervals of cardiac markers based on local Chinese population provides important references to the prevention,diagnosis,efficiency evaluation,and prognosis of CVD.International Federation of Clinical Chemistry and Laboratory Medicine(IFCC)also recommended the establishment of a reference interval applicable to the local population,but in China,systematic studies on establishment of the cardiac markers' reference intervals have not yet been carried out.After establishing cardiac marker reference intervals based on local population,to further address the questions about the individual applicability,intervals the index of individuality(II)and reference change value(RCV)can be calculated by continuously monitoring the biological variation range of the cardiac markers for reference individuals to provide data support for the clinical decision making for CVD.In November 2014,the European Society for Clinical Chemistry and Laboratory Medicine Biology Variations Research Working Group(EFLM WG-BV)initiated a project for the update of biological variation data,jointly organized by six centers located in Europe.However,in the EFLM database released in May 2019,there is no biological variation data of hs-c Tn T and hs-c Tn I.NT pro BNP biological variation data only has a study based on foreign population.On the other hand,there is no biological variation data of hs-c Tn T,hs-c Tn I and NT-pro BNP in China,thus it is not possible to evaluate the application status of individuals and RCV based on the reference interval established by Chinese population.Given that CVD has become a major burden on society,it is generally accepted that prevention is the key to addressing the CVD burdens,but it is still a challenge to identify high-risk populations that may benefit from more aggressive prophylactic treatment.With the increased sensitivity of the cardiac troponin T detecting technology,the detection rate of hs-c Tn T in the natural population was 66%,indicating that subclinical myocardial injury was detected in a high proportion of the community without clinical diagnosed CVD.According to foreign reports,as a noninvasive subclinical myocardial injury marker,hs-c Tn T detection has great potential.For the community population without clinical CVD,hs-c Tn T can help with identifying individuals with high CVD risk.They are in the preclinical phase of CVD and potential changes,thus early recognition can contribute to the early intervention,the progression control,and the prognosis improvement.Currently,there are very limited studies on the related factors regarding the hs-c Tn T elevation and very limited factors were taken into account in such studies.Some factors concluded by foreign studies were failed for the verification on Chinese population;moreover,due to ethnic differences,China's cardiovascular risk factors are different from the Western Caucasians.Therefore,it is remarkably important to determine the Chinese-specific influencing factors of subclinical myocadiac injury with special considerations of the hs-c Tn T distribution,social context characteristcs,physical and laboratory examination results,and disease status.As a conclusion,this study has three research focuses: the cardiac marker reference interval establishment,the hs-c Tn T,hs-c Tn I,and NT-pro BNP biological variation and application,and the hs-c Tn T-related influencing factors study.This study aims to provide standard reference intervals and individual applicability references based on local population and to help with the early CVD identification at prevention level and the early intervention for subclinical myocadiac injury.Methods:Part 1 Establishment and Application of the Reference Intervals of the Cardiac MarkersApplying the stratified cluster sampling protocol,this study takes apparently healthy subjects(With no known CVD or related disease and medication history)in Shenyang area as the screening objects.1848 individuals and 1728 individuals were enrolled in the study of hs-c Tn T and hs-c Tn I screening criteria,respectively.The 99 th percentile URL of hs-c Tn was established by the 2018 AACC / IFCC criteria and the improved selection criteria.932 individuals,932 individuals and 955 individuals were enrolled in the study of CK-MB,Mb and NT pro BNP reference interval.The grouping standard of gender and age is based on the Z-test recommended by ep28-a3 c of CLSI,the Dixon rule is used to exclude outliers,the reference interval and 90% confidence interval of cardiac markers are used by nonparametric method,and the upper reference limit of CK-MB,MB and NT pro BNP is 97.5th percentile.The two subgroups were compared by Mann-Whitney test.Part 2 Biological Variation and Application of hs-c Tn T,hs-c Tn I and NT-pro BNPWith reference to international biological variance studies,this study recruited 25 healthy volunteers in the First Hospital of China Medical University as the research objects.For both the 6-week blood collection cycle and the hs-c Tn T,hs-c Tn I and NT-pro BNP testing process,standardized procedures were implemented to minimize and eliminate pre-assay variability and control analytic variation.The Cochran maximum variance test and the DIXON method were used to exclude the outliers.Nested ANOVA was used to calculate the CVI and CVG and the corresponding formulas were used to calculate II and RCV.The results of biological variation were compared with the Westgard database and EFLM database;the biological variation results were evaluated using the the Westgard database inclusion criteria(PI scores,MM scores,sum of scores,and others).Lastly,this study also compared the biological variances and applications of hs-c Tn T,hs-c Tn I,and NT-pro BNP with domestic and foreign studies.Part 3 Factors Independtly Associated with hs-c Tn T from the General PopulationIn this part of study,the natural population living in Shenyang area was recruited,stratified cluster sampling method was used to select 1562 permanent residents with normal electrocardiogram results,blood measurement and no known cardiovascular diseases,aged above or equal to the age of 18 years.For all included individuals,the following information and results were collected:(1)Background information,including general description,lifestyle,family history,history of disease,(2)Physical examination and laboratory results,including fasting blood glucose,triglyceride,total cholesterol,high density lipoprotein,low Density lipoprotein,creatinine,uric acid,NT-pro BNP,folic acid and hs-c Tn T.According to the newly established reference interval of hs-c Tn T(male: <18ng/L;female: <13ng/L)and the detection limit of 3ng/L,the population was divided into three groups:(1)not detectable(under the detection limit),(2)detectable and(3)elevated,to determine the differences of general status,living habits,family history,physical examination,laboratory tests,and disease status among three groups.Study whether China-PAR can predict myocardial minor injury in three risk groups(Low-risk: 10-year risk < 5%;Mid-risk: 10-year risk 5%-10%;High-risk: 10-year risk > 10%)by hs-c Tn T distribution and level;To investigate the independent risk and protective factors of increased hs-c Tn T,univariate and multivariate linear regression were applied with the logarithm of hs-c Tn T as the dependent variable(hs-c Tn T results under detectable range were assigned with value of 1.5ng/L);single factor and multi-factor logistic regression analysis were applied with the hs-c Tn T increase as the dependent variable.Results:Part 1 Establishment and Application of the Reference Intervals of the Cardiac Markers1.The 99 th percentile URLs of hs-c Tn T under different selection procedures If the 2018 AACC/IFCC criteria was applied,99 th percentile URLs(90% Confidence Interval)of hs-c Tn T male and female were 19(17-20)ng/L and 16(15-17)ng/L respectively.If added a single supplementary selection criteria,hypertension or overweight and obesity or dyslipidemia,99 th percentile URLs of hs-c Tn T male and female remained the same.If the improved selection criteria was applied,99 th percentile URLs(90% Confidence Interval)of hs-c Tn T male and female were 18(14-24)ng/L and 13(11-16)ng/L respectively.2.The 99 th percentile URLs of hs-c Tn I under different selection procedures If the 2018 AACC/IFCC criteria was applied,99 th percentile URLs(90% Confidence Interval)of hs-c Tn I male and female were 18(13-19)ng/L and 10(8-14)ng/L respectively.If added a single supplementary selection criteria,hypertension or overweight and obesity or dyslipidemia,99 th percentile URLs of hs-c Tn I male under the exclusion of dyslipidemia and 99 th percentile URLs of hs-c Tn I female under the exclusion of overweight and obesity will remain unchanged,the rest will be reduced.If the improved selection criteria was applied,99 th percentile URLs(90% Confidence Interval)of hs-c Tn T male and female were 17(11-19)ng/L and 7(6-11)ng/L respectively.3.Distribution of hs-c Tn T values by age and gender under improved selection criteriaIn each age group,the URL of the 25 th,median,75 th and 99 th percentile values of hs-c Tn male were higher than that of hs-c Tn female(p = 0.037 for hs-c Tn I over 50,p < 0.001 for the other groups),and the age group over 50 was higher than the age group under 50.4.Comparison of the reference interval results of other three cardiac markers with the data in the reagent manualDifferent from the gender grouping standard in the reagent specification,the reference interval of CK-MB is grouped by age: less than or equal to 40 years old CK-MB <2.49ng/m L and greater than the age of 40 CK-MB<5.41ng/m L.The reference interval of Mb is grouped by gender: 21ng/m L<Mb male <60.38ng/m L,21ng/m L <Mb female <41.99ng/m L.It is the same grouping standard of the reagent specification,but lower than the value of the reagent specification.Different from the uniform grouping standard in the reagent specification,the reference interval of NT-pro BNP is grouped by age: ?60 years old NT-pro BNP<114ng/L and >60 years NT-pro BNP<248ng/L.Part 2 Biological Variation and Application of hs-c Tn T,hs-c Tn I and NT-pro BNP1.Biological variation results for hs-c Tn T,hs-c Tn I and NT-pro BNP.Results of both hs-c Tn I(CVI 5.72%,CVG 7.57%)and hs-c Tn T(CVI 16.89%,CVG 14.33%)were smaller than those of the Westgard database;the corresponding results of hs-c Tn T were greater than those of hs-c Tn I;The results of NT-pro BNP(CVI 11.03%,CVG 17.85%)were essentially the same as those of the Westgard database,but data smaller than EFLM database(CVI and CVG were 25.1% and 54.0%,respectively).2.The quality evaluation of the biological variation resultsThis study was devoted to the study of biological variants,incorporated into healthy adult data,and the median of all data was generated together.The PI score of hs-c Tn I was 1;the PI score of hs-c Tn T and NT-pro BNP was 2.MM scores are all 4.Hs-c Tn I has sum-score of 5,hs-c Tn T and NT-pro BNP has sum-score of 6.3.The application results(RCV and II)of the hs-c Tn T,hs-c Tn I and NT-pro BNP biological variationHs-c Tn I(II 0.76),hs-c Tn T(II 1.18)and NT-pro BNP(II 0.62)were all between 0.6 and 1.4.The RCV of hs-c Tn I is between-16.38% and 19.6%;the RCV of hs-c Tn T is between-38.06% and 61.5%;the RCV of NT-pro BNP is between-26.32% and 35.7%.The RCV of hs-c Tn T is greater than that of hs-c Tn I.4.Comparison of hs-c Tn T,hs-c Tn I and NT-pro BNP biological variation and application(RCV and II)with domestic and foreign studies.Hs-c Tn T(CVG 14.33%)?hs-c Tn I(CVI 5.72%,CVG 7.57%)and RCV(between-16.38% and 19.6%)were less than results of all other studies.Other results of this study were all resided among the different studies.Part 3 Factors Independently Associated with hs-c Tn T from the General Population1.The relationship between the level and distribution of hs-c Tn T and China-PARThere was no statistical difference in the proportion and level of hs-c Tn T in the low-risk,intermediate-risk,and high-risk groups(p > 0.05).2.Univariate and multivariate linear regression of hs-c Tn T levelsAccording to the results of the multivariant logistic regression analysis with the logarithm of hs-c Tn T value as the dependent variable(hs-c Tn T results under the detectable range were assigned with the value of 1.5ng/L),age,rural area,educational level,exercise,hyperuricemia,hyperglycemia,renal dysfunction,obesity and NT-pro BNP were all found to be significantly positively correlated to the level of logarithm of hs-c Tn T(p < 0.05).Female gender,sedentary time,sleeping time and folic acid were all found to be significantly negatively correlated to the level of logarithm of hs-c Tn T(p < 0.05).3.Multivariate logistic regression analysis of the influencing factors on elevated hs-c Tn TThe multivariate logistic regression analysis with the hs-c Tn T increase as the dependent variable implied that age(a OR 1.124,95% CI 1.082,1.168),rural area(a OR 3.083,95% CI 1.268,7.496)and hyperglycemia(a OR 4.574,95% CI 1.660,12.604)were all risk factors for the hs-c Tn T elevation.Sedentary time(less than 1 hour per day)(a OR 0.125,95% CI 0.022,0.707)was the protective factor for the hs-c Tn T elevation.Conclutions:1.Compared with the 2018 AACC / IFCC criteria,the improved selection criteria(further excluding hypertension,overweight,obesity and dyslipidemia through questionnaire survey,physical examination and laboratory screening)can avoid overestimating the 99 th percentile URL of hs-c Tn;the single screening standard has different effects on hs-c Tn T and hs-c Tn I;2.The reference intervals of Chinese cardiac markers(hs-c Tn T,hs-c Tn I,NT pro BNP,CK-MB and Mb)established by the improved selection criteria is quite different from the data in the reagent specification;hs-c Tn T,hs-c Tn I and MB need to set reference interval according to gender;NT-pro BNP and CK-MB need to set reference interval according to age;3.The research quality evaluation show the biological variation results reported in this study have met the Westgard database and EFLM database standard.The biological variation of hs-c Tn T,hs-c Tn I,and NT-pro BNP among Chinese population was different from that among foreign population;4.While the diagnosis and treatment of CVD,for hs-c Tn T,hs-c Tn I and NT-pro BNP,the clinicians need to take into account the reference intervals and the continuous RCV detection.The RCV variation within the reference range should be considered physiological fluctuation.The disease status differenciation,efficiency evaluation,and prognosis of CVD should all exceed this range.The biological variation and RCV of hs-c Tn T and hs-c Tn I are both different.Therefore,while continuous monitoring and evaluating the patients' hs-c Tn T and hs-c Tn I levels,one should also consider the difference between these two analytes.5.China-PAR can not predict myocardial micro injury in CVD Free population.In addition,there is still an small increase of hs-c Tn T in the natural population without CVD.Age,rural area and hyperglycemia are all independent risk factors for hs-c Tn T elevation.Sedentary time(less than 1 hour per day)is the only independent protective factor for hs-c Tn T elevation.