Short-term increases of plasma cardiac troponin I are better evaluated by comparison with the reference change value

Materials and methods: Patients admitted for chest pain (N = 103) were evaluated retrospectively on the basis of two consecutive cardiac troponin-I (cTn-I) tests. The second test levels exceeding the “Critical Reference Change Value” (CrRCV), a quantity calculated on the basis of the 9 rst result and the RCV of cTn-I, were considered particularly relevant. Clinical cases were analysed matching the concentration change (signi9 cative or not) with acute coronary syndrome (clinically con9 rmed or not). Healthy individuals (N = 70) results and internal quality control results were evaluated for the calculation of, respectively, the biological and the analytical variation of plasma cTn-I.


Introduction
Cardiac troponin concentration is the most useful biomarker for suspected acute coronary syndrome (ACS) management in (ED).The troponin assay confi guration, featuring a combination of antibodies against the heart-specifi c and stable region of the molecule, determines their clinical performance.However, the interpretation of test results requires not only a comparison with a reference interval or threshold value, but also a comparison between consecutive measurements (1).International recommendations of Cardiology Society and Laboratory Medicine established that the Tn-I decision limit should be at the 99 th percentile value of a reference group with a total analytical im-precision (as coeffi cient of variation) ≤ 10 % (2).Actually, few studies have considered a longitudinal comparison, as estimated by the change between two consecutive troponin tests.We investigated this specifi c point by analysing the variations of troponin-I in two consecutive tests from the same patient, as obtained in the laboratory routine practice.
This work aimed at: a) estimating the biological and analytical variation of plasma cardiac troponin I (cTn-I) measurement for a specifi c analytical system, to be used for the reference change value (RCV) calculation; b) assessing the diagnostic accu-Introcaso G. et al.
cTn-I measurements and RCV racy of the RCV of cTn-I for diagnosing the ACS in patients with second elevation of cTn-I after 4-6 hours.

Patients and analytical methods
From the laboratory database we collected data from 103 patients (age between 31 and 93 years, median age 72) admitted to the cardiological ED in a period of 9 months.Patients inclusion criteria were based on the cTn-I increases in the ED with the fi rst value of the cTn-I < 0. Biological variation 70 healthy individuals (41 women and 29 men; healthy state assessed by medical evaluation and laboratory tests) were enrolled to evaluate withinsubject (CVw) and between-subject (CVbt) biological variation of cardiac troponin-I.Ten subjects out of 70 were retested during the period of study, under routine analytical conditions and according to suggested protocol, to estimate the CVw (3,4).During a period of six months, three blood samples, randomly in diff erent working day, were drawn and each subject assayed.According to preliminary data, we reputed suffi ciently accurate the protocol for CVw evaluation with 10 subjects tested (5).
The cTn-I CVw, as well as the cTn-I CVbt of the population, was calculated as coeffi cient of variation (CV) obtained by the cTn-I measurements of the healthy subjects (Table 1).The CV was calculated as the ratio of the standard deviation to the mean of all the cTn-I measurements.

Analytical variation
The analytical variation was calculated as the coeffi cient of variation observed during 9 months of internal quality control of the analytical system, at three concentration levels, and including control charts monitoring and application of control rules (Table 1).In order to further evaluate the analytical imprecision of the analyser, we have performed an imprecision profi le study (Figure 1).A pool of 32 plasma samples were used to prepare 7 pools with cTn-I concentrations ranging from 0.02 to 6 µg/L.They were stored at -30 °C until measurement, when they were thawed, equilibrated to room

Introcaso G. et al. cTn-I measurements and RCV
temperature, and centrifuged before measurement.
We then performed repeated measureme nts of each aliquot per day in a period of about 3 months.Finally, the total coeffi cient of variation was calculated for each cTn-I concentration (Table 1).

Preanalytical conditions
Blood samples with heparin as anticoagulant were collected, centrifuged at 2500 x g for 10 minutes, and analysed freshly.Adequate fi lling of the collection tube and immediate mixing after collection were assured.Haemolysed samples with free haemoglobin > 500 mg/dL were excluded.

RCV and the cTn-I critical value RCV
The RCV for two consecutive values of cTn-I was calculated according to Fraser and Harris' formula (6,7): where Z is the z-statistic equal to 1.96 at 95% statistical signifi cance, CVw is the within-subject biological variation and CVa is the analytical variation.
In order to assess the signifi cance of possible differences between two consecutive results from the same patient, we have introduced "critical value of troponin RCV" (CrRCV) calculated as follows: where CV and CrRCV are expressed in unit of concentration (µg/L).This approach allows an immediate evaluation of the signifi cance of any diff erence between two measured consecutive values of cTn-I: when the second concentration value is higher than the CrRCV, any observed increase is statistically signifi cant.

Results
We found a within-subject biological variation CVw equal to 86% as coeffi cient of variation of the 30 cTn-I measurements obtained from 10 healthy subjects; the analytical variation was 4.4%, at the concentration level of 0.4 µg/L and for higher levels (see Table 1); from these we estimated a RCV equal to 240%.This result was due to the high analytical variation of cTn-I measurement at the low concentrations, close to the analytical detection limit 0.01 µg/L, of the majority of healthy individuals.Consequently, we justifi ed an overestimation of the cTn-I biological variation measure.The cTn-I imprecision profi le (Figure 1) showed that at 0.02 µg/L the analytical variation as total CV was 32%, while, the 10% of CV was reached at 0.07 µg/L, close to the decision limit for myocardial injury (equal to 0.05 µg/L, corresponding to the 99 th percentile of a reference population).
The increased cTn-I concentrations for patients admitted to ED for the suspect of ACS and confi rmed through clinical and angiographic data were clinically signifi cant.
The initial concentration value at admission to ED, for patients discharged after clinical evaluation, was 0.015 µg/L (median value) with a maximum of 0.08 µg/L.This means that some cTn-I values clinically negative can fall close to the decision limit of 0.05 µg/L in the suspect of ACS.
From the laboratory database we have selected data with the fi rst cTn-I result ≤ 0.1 µg/L, then a comparison of the second result with the CrRCV was assessed.As derived quantities of Chi square analysis, we have estimated the cTn-I RCV negative predictive value (NPV) equal to 88% with confidence interval at 95% (CI = 82-92%).The positive predictive value (PPV) for cTn-I RCV was 52% with (CI = 37-64%).The sensitivity was 62%, (CI = 44-77%) and the specifi city 83%, (CI = 77-87%).The application of cTn-I RCV critical value to the two consecutive results from 103 patients admitted to ED is shown in Table 3.We found that the cTn-I RCV was eff ectiveness for the detection of some ACS cases; conversely, it can exclude, with high probability, the not signifi cant cTn-I increases.We found that patients suff ering from severe coronaropathies exhibited a high percentage of signifi cant increase of cTn-I between two consecutive results (Table 2).
The most consistent result concerns the cTn-I increase of the group of patients discharged.We have shown that many patients with low cTn-I increases and not exceeding the cTn-I RCV were discharged after clinical evaluation (Table 3).The difference between cTn-I RCV values for patients admitted to hospital with a fi nal diagnosis and values for patients discharged from ED was statistically signifi cant (Chi square P < 0.001).

Discussion
Cardiac troponins I or T are commonly used in clinical laboratory as biomarkers of myocardial necrosis.In an experimental comparison study, the clinical performance of Accu troponin-I (cTn-I) assay (Beckman Coulter) has been assessed (8).The AccuTn-I assay, based on a pair of monoclonal anti-

Introcaso G. et al. cTn-I measurements and RCV
bodies against epitopes close to the NH 2 terminus (epitopes 24-40 and 41-49), actually shows a good sensitivity and specifi city for acute coronary syndrome.Commercial assays and analytical systems are available from the diagnostic manufacturers with improved test sensitivity and analytical reliability; however, it is well understood that the future generations of assays for cardiac troponin should improve the sensitivity of biomarkers detection (9,10).In the last period, several studies in progress have the aim to assess diagnostic assays with ultrasensitive cTnI, controlling the analytical specifi city.The technology-dependent sensitivity limits or unrecognizable interferences might cause misleading laboratory report (11).The interpretation of laboratory tests remains important to evaluate and quantify the myocardial injury in order to improve the prognosis of the patients (12).In particular, the longitudinal comparison of biomarker results in serial determinations is not presently given enough attention in clinical laboratory routine as well as the reliable statistical measure of reference population (13,14).In the present observational retrospective study, we have applied the CrRCV, a new RCV derived parameter, to interpret the second of two consecutive cTn-I measurement results in the evaluation of clinical cases in ED.To investigate the clinical eff ectiveness of cTn-I RCV, calculated in our laboratory, we have analysed the retrospective cTn-I increases in diff erent cardiological pathologies.We have considered low elevations of cTn-I results even with concentrations be-low the 99 th percentile of the reference population.In fact, recent studies have demonstrated that minor elevations of cardiac troponins are clinically signifi cant for cardiovascular events as well as elevated troponin levels (15,16).
Patients admitted for chest pain to ED revealed that many cTn-I increases can have a casual fl uctuation that could determine a misclassifi cation of patients.In fact, the cTn-I RCV showed a high ne-   gative predictive value with a statistically significant association with the exclusion of ACS in patients in ED (see Table 3).However, in AMI or severe coronaropathy cases, the short-term increased cTn-I concentration, greater than cTn-I RCV, could better evaluate the biochemical event of the myocardial necrosis.On the other hand, the calculation methodology of cTn-I RCV introduces a tool to quantify the imprecision and analytical noise of immunoassay technique for low troponin concentrations.According to our fi nding, a multicenter study conducted on the commercially available assays has demonstrated, with the same experimental protocol, a high cTn-I imprecision at low concentration ranges (17).These investigations suggest that the analytical and biological variability of cardiac troponin-I should be quantifi ed, and the reference change value should be applied as indicator of the signifi cance of the change between two consecutive values (18).Nevertheless, our laboratory results suggest that they may not be applied extensively (without extrapolation to the general population), but they are valuable and deserve further investigation.The cTn-I RCV calculated, could be applied in medical routine only for low cTn-I levels (< 0.1 µg/L), and for a specifi c immunoassay technique, because of its intrinsic imprecision (17).
In the evaluation of cTn-I levels around the decision limit of 0.05 µg/L, non signifi cant plasma variations could be detected and interpreted by comparison with the cTn-I RCV.In fact, the high NPV of cTn-I RCV suggests its possible use to aid the rule out of patients in ED with minor not specifi c cTn-I elevations.We focused on the features of the immunochemical method that determine a high analytical imprecision of biomarker, close to the detection limit of 0.01 µg/L, with a contribution to high values of biological variability and RCV.We recognised the limitations of our study, also, in regard of the RCV calculation and sensitivity of the analytical method routinely used.An additional adverse eff ect of low sensitivity of method makes the RCV estimates method-dependent.In practice, a lower detection limit of cTn-I assay could improve the within-subject biological variation estimation, which the RCV calculation is based upon.In conclusion, we admitted limitations both in regard of the homogeneous population of the clinical cases due to a lack of baseline comparability, and to the poor evidences to distinguish among the possible forms of association.However, the diagnostic accuracy was done retrospectively and surely might have introduced a certain amount of overestimation.In any case, a reference change applied to a longitudinal analysis of cardiac biomarkers remains a challenge that requires further clinical and prospective studies.

FIGURE 1 .
FIGURE 1.Imprecision profi le of cTn-I measurement obtained by our analytical system.For concentrations close to the analytical sensitivity limit (0.01 µg/L) the total CV becomes very high.

TABLE 1 .
Sources of variability of the cTn-I measurements for cTn-I RCV calculation.It's very high variation close to the detection limit of 0.01 µg/L.

TABLE 2 .
Amount of plasma cTn-I increases in pair of consecutive measurements.Figures are the concentrations and cTn-I increases expressed as median value and IQR.Plasma cTn-I increase, 2nd over 1st sample.

TABLE 3 .
Cases with increased plasma cTn-I concentration in the