Cardiac troponin I (cTnI) plays a fundamental role in diagnosing myocardial injury [1]. The implementation of cTnI assays is critical in identifying patients at high risk of cardiovascular disease and assists in the risk stratification of patients undergoing major non-cardiac surgeries [1,2].

The Universal Definition of Myocardial Infarction recommends the use of high-sensitivity cardiac troponin (hs-cTn) assays, with the 99th percentile upper reference limit (URL) as the diagnostic threshold for myocardial infarction [1]. Additionally, the current guidelines from the International Federation of Clinical Chemistry and Laboratory Medicine specify that high-sensitivity assays must exhibit a 10% coefficient of variation (CV) at the 99th percentile URL for a healthy population and detect cTn above the limit of detection (LoD) in 50% of the healthy population [3]. These guidelines underscore the importance of precise and sensitive cTnI detection. In the late 1980s, the troponin assays could detect cTn at ng/mL levels. On the contrary, recent hs-cTn assays can detect cTn at pg/mL levels, even enabling the identification of minimal myocardial injury [4].

Researchers have explored various methods and are actively engaging in the development of ultrasensitive cTnI assay kits. Among these methods, the chemiluminescence immunoassay (CLIA) stands out because of its high sensitivity, rapid reaction, simple instrumentation, and wide dynamic range [5].

Our study evaluates the performance of the newly developed CLIA, ALFIS (Boditech Med Inc., Chuncheon, Korea). Additionally, we assessed the impact of various anticoagulants, as this new immunoassay accommodates both serum and plasma samples.

We evaluated the analytical performance of the ALFIS cTnI assay according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. The study was approved by the Institutional Review Board of Seoul St. Mary’s Hospital (IRB no., KC20TNSI0585), and the board waived informed consent because this study utilized quality materials prepared by the manufacturer and residual serum samples.

1. Description of the ALFIS cTnI assay

The ALFIS assay is conducted using a pair of antibodies specific to TnI. A magnetic bead is coupled to one of the antibody pairs, and an alkaline phosphatase (ALP) is coupled to the other antibody to perform a sandwich reaction for TnI, forming a magnetic bead-antigen-antibody-ALP complex. The complex is captured and transferred to the wash well to remove non-reactive substances to increase the signal. After TnI is captured and purified, it is transferred to the measurement well containing the chemiluminescent substance CDP-Star (Invitrogen, T2214; Thermo Fisher Scientific, Waltham, MA, USA), and the signal is detected and converted to concentration using a formula. Since it operates in a sandwich format rather than a competitive format, the signal increases proportionally with the concentration of TnI (Fig. 1).

Figure 1. The principle of troponin I (TnI) detection via the ALFIS cardiac TnI assay system. Abbreviation: ALP, alkaline phosphatase.

The manufacturer provided seven levels of quality materials of varying ratios of human Troponin Complex (I-T-C) (Hytest) stocks and horse serum (Gibco; Thermo Fisher Scientific) diluent mixtures. The TnI concentrations of each level were determined to be 0, 30, 60, 100, 450, 880, and 4,220 ng/L by the reference method, Access 2 (Beckman Coulter, Brea, CA, USA). Each material was measured 5 times by the test product. A linear fit model obtained the regression equation (x, measured concentration; y, expected concentration).

2. Imprecision

Imprecision was evaluated following the CLSI guideline EP15-A3 [6]. Three concentrations of quality materials (230 ng/L, 940 ng/L, and 7,500 ng/L by Access) were measured in duplicates twice daily and for 20 consecutive working days. The standard deviation and CV were calculated to assess the repeatability and within-laboratory imprecision at each level.

3. Analytical sensitivity

The limit of blank (LoB), LoD, and limit of quantification (LoQ) were determined according to the CLSI EP17-A protocol [7]. The LoB was assayed using four blank samples made from horse samples, and all measured less than 20 ng/L by the reference assay. The reference assay assayed the LoD with four samples with an expected concentration of <10 to 40 ng/L. The low-concentration samples were measured in five replicates per day for 3 days. The LoQ was determined as the lowest concentration with CV ≤10% when five samples of low TnI concentrations from 10 to 50 ng/L were tested in four replicates a day for 3 days.

4. Correlation study

The cTnI concentrations of 41 residual patient serum samples were compared between the ALFIS and Atellica IM TnIH (Siemens Healthineers, Erlangen, Germany) on the Atellica IM analyzer according to the CLSI guideline EP9 [8]. Pearson correlation and Deming regression analyses evaluated the correlation between the assay values.

5. Cross-reactivity and interference

To examine the potential cross-reactivity with other cardiac biomarkers, the test product was assessed for its ability to detect TnI-negative serum samples containing other compounds, such as skeletal troponin I (250 ng/mL), cardiac troponin C (250 ng/mL), cardiac troponin T (125 ng/mL), CK-MB (60 ng/mL), myoglobin (1,000 ng/mL), myosin (1,000 ng/mL), tropomyosin (1,000 ng/mL), actin (1,000 ng/mL), N-terminal pro-brain natriuretic peptide (NT-proBNP) (1,000 ng/mL), and D-dimer (1,000 ng/mL). Cross-reactivity was considered significant if the percent recovery deviated by more than 10%.

Following the CLSI EP7-A2 guideline [9], we evaluated the interference effect of the hemoglobin, bilirubin, D-glucose, L-ascorbic acid, cholesterol, and triglyceride mixture on the accuracy of the test product. Moreover, commonly used anticoagulants, such as ethylenediaminetetraacetic acid (EDTA), sodium citrate, and heparin, were examined for their potential interference. Interference was considered significant when the percent recovery deviated by more than 10%.

6. Statistical analysis

Statistical analysis was performed using Microsoft Office Excel (Microsoft Corp., Redmond, WA, USA) software and Prism ver. 10.0.3 for Windows (GraphPad, San Diego, CA, USA).

1. Imprecision

Seven levels of quality materials were used to calculate the regression equation. The regression equation relating measured (y) to expected values (x) was determined as y=2.577x+134.5 based on a linear fit (R²=0.9985) (Fig. 2). The repeatability, as indicated by CV, ranged from 4.8% to 6.3%. The CV values ranged from 4.7% to 7.4% for within-laboratory imprecision assessment. All the CV values met the desirable imprecision specification of 10% at all concentrations (Table 1).

Table 1 . Precision of the ALFIS cTnI assay.

Material	Repeatability			Within-laboratory imprecision
	Mean±SD (ng/L)	CV (%)		Mean±SD (ng/L)	CV (%)
230 ng/L	230±0.015	6.3		230±0.017	7.4
940 ng/L	950±0.056	5.9		950±0.065	6.8
7,500 ng/L	7,550±0.363	4.8		7,530±0.351	4.7

Abbreviations: SD, standard deviation; CV, coefficient of variation..

Figure 2. Linear regression curve of ALFIS cardiac troponin I assay. The values assigned by reference method are on x-axis and the luminous intensity measured by the test product are presented on y-axis. Abbreviation: RLU, relative light units.

2. Analytical sensitivity

The LoB and LoD values were calculated non-parametrically. The calculated LoB and LoD values were 4 ng/L and 10 ng/L, respectively. The 10% CV was at 30 ng/L.

3. Correlation study

The cTnI levels were measured by the ALFIS and Atellica IM assays in 41 patient samples. The comparison was performed on 37 patient samples after excluding three samples measured below the LoD of 10 ng/L and one outlier sample of 966 ng/L by ALFIS. The included sample concentrations of TnI range from 15 to 405 ng/L by ALFIS. The ALFIS cTnI revealed a Pearson correlation coefficient of 0.8478 (P<0.0001), and the equation of the Deming regression analysis was ALFIS=0.5229×(Atellica)–35.21 (Fig. 3A). Based on the Bland-Altman analysis, the mean difference between the two methods was –82.9, with 95% limits of agreement of –16.1 to –149.7 (Fig. 3B).

Figure 3. Scatter plot for method comparison. (A) Correlation between Atellica IM TnIH (x-axis) and ALFIS (y-axis) for clinical serum samples. The line represents the equation of Deming regression. Pearson coefficient (r) and P-value were given at the upper left corner. (B) Bland-Altman analysis between Atellica and ALFIS. The mean differences between AFLIS and Atellica (y-axis) are plotted against the averages of the two methods (x-axis). The horizontal dotted lines indicate 95% limits of agreement. Abbreviation: RLU, relative light units.

4. Cross-reactivity and interference

Overall, the test product did not demonstrate significant cross-reactivity with the tested cardiac biomarkers, as shown in Fig. 4A. The only exception was NT-proBNP with a cTnI value of 230 ng/L, for which the percent recovery increased slightly to 112.8%. Similarly, most test results were unaffected by the addition of the possible interferents, as described in Fig. 4B. As an exception to this trend, the percent recovery was 78.7% to 78.8% with EDTA, resulting in more than 20% negative interference.

Figure 4. Cross-reactivity (A) and interference (B) study of ALFIS cardiac troponin I assay. The x-axes represent cardiac biomarkers (A) and possible interferents (B), while the y-axis represents the recovery (%). Horizontal dotted lines indicate 100%±10% recovery (%). Values within the lines indicate nonsignificant response. Abbreviations: L, low level (230 ng/L TnI); M, medium level (940 ng/L TnI); H, high level (7,500 ng/L TnI); sTnI, skeletal troponin I; cTnC, cardiac troponin C; cTnT, cardiac troponin T; CK-MB, creatinine kinase muscle brain; NT-proBNP, N-terminal pro-B type natriuretic peptide; Hb, hemoglobin; Bil, bilirubin; Glc, D-glucose; Vit.C, L-ascorbic acid; Chol, cholesterol; TG, triglyceride mixture; EDTA, ethylene-diamine-tetraacetic acid; citrate, sodium citrate.

Previous studies have ascertained that cTnI is a vital biomarker of cardiac ischemia and can help clinicians in detecting and diagnosing acute myocardial infarction and heart failure and in the prognosis of adverse cardiac outcomes following non-cardiac surgery [2,10,11]. The above-mentioned clinical utility has much to do with the high-sensitivity detection of cTnI; moreover, several studies have focused on developing ways to accurately and sensitively measure cTnI while trying to make it more cost-effective. Compared to the commonly used rapid methods, the chemiluminescent approach differs significantly in that it allows for a quantitative analysis of the amount of TnI in the sample rather than just a qualitative assessment. In comparison to point-of-care (POC) devices using fluorescence analysis, the chemiluminescence method excels by operating in a low-background environment and recognizing signals generated by its inherent chemiluminescence, thereby providing a considerable advantage in sensitivity.

Our results indicate that the imprecision of the assay meets the pre-specified quality requirement of CV <10% at all measured concentrations. However, the analytical sensitivity of ALFIS needs to be further improved as the LoD and LoQ of the test assay fall behind those of the previously developed Access 2 kit, 0.63 ng/L and 6.1 ng/L, respectively [3]. To be used as a POC device for diagnosing acute myocardial injury, special attention is required to improve the LoQ of the test assay to the 6 ng/L level suggested for identifying low-risk individuals [12]. Additionally, the 99th percentile URL should be further examined.

The correlation study using clinical samples showed a relatively low correlation between ALFIS and Atellica IM TnIH, a high-sensitivity cTnI assay used in central laboratories. A negative systematic bias was observed. To improve the accuracy of the test assay, the proper management of calibration adjustments is necessary.

Our findings suggest the need to improve the specificity of ALFIS. Significant false-positive reactions to other cardiac biomarkers were not detected during the cross-reactivity evaluation, except for a slightly increased recovery with NT-proBNP. It is crucial to address potential cross-reactivity issues related to NT-proBNP. We evaluated the effect of potential interferents, which revealed no significant effect of hemoglobin, bilirubin, and other compounds biologically present in the human sample. This is in line with the previous findings that bilirubin <45 mg/dL, hemoglobin <560 mg/dL, triglyceride <3,000 mg/dL, and total cholesterol <2,000 mg/dL did not exert any interference [4]. Since the kit can be used for both serum and plasma samples, we tested the effect of anticoagulants and observed negative interference with EDTA (>20%); however, no such effect was observed with other anticoagulants, such as sodium citrate and heparin.

In summary, the overall performance of the ALFIS cTnI assay was assessed. The proper address of the previously mentioned issues enables the utilization of this assay in various clinical settings, providing an additional option for laboratories with limited space and cost.

This research was supported by the MOTIE (Ministry of Trade, Industry, and Energy) in Korea under the World Class 300 Project (R&D) (P0012998, development of an automated liquid-based diagnostic system using high-sensitivity chemiluminescence for the diagnosis of cardiovascular and other biomarkers), supervised by the Korea Institute for Advancement of Technology (KIAT).