Analytical and clinical evaluation of the light‐initiated chemiluminescent assays for measurement of human thyroid hormones

Abstract Background Light‐initiated chemiluminescent assay (LiCA) is a new homogeneous immunoassay. The aim of this study was to evaluate the analytical and clinical performance of the assays for the detection of thyroid hormones based on the fully automated LiCA 800 analyzer. Methods Analytical validations of the LiCA thyroid assays (TSH, FT3, FT4, T3, and T4) included precision, linearity, analytical sensitivity, interference, and method comparison applying the protocols of the Clinical and Laboratory Standards Institute (CLSI). The diagnostic performance was assessed by the receiver operating characteristic (ROC) curve analysis with different assay schemes for the diagnosis of hyperthyroidism and hypothyroidism. Results Within‐run and within‐lab precisions (%CV) of the five assays ranged from 1.06 to 6.40% at all concentrations evaluated. A satisfactory linearity was verified over the entire measuring range for TSH, T3, and T4 (R > 0.99, change in recovery <10%, p = 0.000 all). Paired‐comparison measurements presented a comparable assay for each of the five assays (R > 0.96, median bias <5%, p < 0.0001 all) between LiCA and Cobas across three institutes. The diagnostic accuracy of the LiCA assays for hyperthyroidism or hypothyroidism was quantified by the areas under curves (AUC) as 0.925 or 0.832 with the five‐assay panel (TSH, FT3, FT4, T3, and T4) and as 0.921 or 0.811 with the three‐assay panel (TSH, FT3, and FT4), respectively. No significant difference was found between the AUC of LiCA and that of DxI, Cobas, or Centaur (p > 0.3 all). Conclusion LiCA 800 provides a precise and high‐throughput immunoassay platform for detection of thyroid hormones. It is acceptable for clinical use.

With overt advantages on high sensitivity and specificity, full automation, high-throughput, and non-radioactive contamination, chemiluminescent immunoassay (CLIA) is gradually taking the leading position for the thyroid tests in clinical laboratory. 3 Due to lack of harmonization on the international level of reference materials, detection of thyroid assays could be variant from different platforms based on different methodologies. Thereby, a performance validation is necessary before a new assay could be admitted in clinical application.
In this study, we introduced a new homogeneous light-initiated chemiluminescent assay (LiCA ® ) that was derived from the luminescent oxygen-channeling immuno-technology described by Ullman et al. 4,5 The key analytical characteristics of the five thyroid assays (TSH, FT3, FT4, T3, and T4) were assessed following the guidelines of the Clinical and Laboratory Standards Institute (CLSI). In addition, the diagnostic performance was analyzed by the receiver operating characteristic (ROC) curve with different assay schemes for the diagnosis of hyperthyroidism and hypothyroidism.

| Study samples
The evaluation study of the five thyroid assays (TSH, FT3, FT4, T3, and T4) was conducted at the clinical laboratory of Peking University international Hospital. Additional method comparison between LiCA and Cobas was validated at other two institutes in China. All samples were collected from residual and de-identified patient sera within 2 h after clinical routine thyroid assays. We selected specimens based on serum quality, volume, and analyte concentrations.
Subjects with visible icterus, lipemia, or hemolysis were excluded.
Unless otherwise noted, specimens were stored at −20°C for no longer than 30 days with one freeze-thaw cycle in this study.

| LiCA ® thyroid assays
The LiCA ® thyroid assay (Chemclin Diagnostics, Beijing, China) uses two nanobeads (sensitizer and emission) to bridge a fully homogeneous immunoassay. When the antigen-antibody complex is formed after incubation, the distance between two beads is less than 200 nm. Singlet oxygen generated from the sensitizer by light at 680 nm diffuses to the emission bead across the complex, thus triggering a chemiluminescent reaction. In contrast, no emission occurs if there is no antigen-antibody reaction, as a longer distance (>200 nm) blocks energy transmission. Therefore, no-wash is necessary to separate the immune complex from free components in the cuvette, and thus, this test delivers a fast and stable measurement. [6][7][8] The LiCA 800 analyzer used in this study is a fully automated, random-access, and high-throughput (600 tests per hour) immunoassay platform.
Time to the first report for each of the LiCA thyroid assays (TSH, FT3, FT4, T3, and T4) is 32 min. The assay specifications have been summarized in Table 1.

| Precision study
A precision study was performed for each of the LiCA thyroid assays in accordance with the CLSI EP15-A3 protocol. 9 Repeatability (within-run) and within-laboratory (intermediate) imprecision (coefficient of variation, CV) were determined using three levels of pooled human sera. 25 measurements were collected for each sample in 5 runs over 5 days.

| Analytical sensitivity study
We followed the CLSI EP17-A2 protocol 11 to examine the limit of blank (LoB), limit of detection (LoD), and limit of quantitation (LoQ) for the LiCA thyroid assays. The LoB experiment was performed with the zero-point manufacturer's calibrator. LoD and LoQ were determined using serial low levels of pooled sera. Each sample was aliquoted into 20 tubes. Triplicate measurements were performed during 20 runs over 5 days (n = 60).

| Interference study
To evaluate for possible interference of hemolysis, lipemia, and icterus in the LiCA thyroid assays, three levels of patient serum samples were spiked according to manufacturer's claims with interferents that included hemoglobin (2.5 g/L), triglycerides (5.65 mmol/L), and bilirubin (171 μmol/L), respectively. Each sample was measured in duplicate, and percent recovery was calculated for the evaluation of interference.

| Method comparison study
A three-site comparison study was carried out for the LiCA and Cobas thyroid assays following the guideline of CLSI EP9-A2. 12 Serum specimens were collected from randomly selected patients with analyte concentrations across the analytical measuring range.
All samples were assayed on the same day with the Cobas e602 (Roche Diagnostics) and LiCA 800 analyzers. Measuring agreement was evaluated by linear regression and bias plot.

| Precision
As shown in Table 2, the within-run CV% of each assay ranged from 1.06% to 5.63% and the within-lab CV% from 1.55% to 6.40%, across low, middle, and high levels of pooled human sera.
Our findings revealed that LiCA provided a good precision in thyroid assays.

| Linearity
The The percent recovery obtained for each dilution was 96.4%-106.2% for TSH, 94.1%-107.4% for T3, and 95.6%-107.8% for T4, respectively. The hypothesis of a linear fit was accepted for each of the assays.

| Analytical sensitivity
The results of LoB, LoD, and LoQ (20% CV) for the LiCA thyroid assays were displayed in Table 3. Our observations agreed with the manufacturer's claims.

| Interferences
No analytical interference was observed for the LiCA thyroid assays in the presence of hemoglobin (2.5 g/L), triglycerides (5.65 mmol/L), and bilirubin (171 μmol/L) as claimed by the manufacturer ( Table 4).
The percent recovery for each of the assays was determined to be between 92.5% and 108.0% across low, middle, and high levels of serum samples. Abbreviations: LoB, limit of blank; LoD, limit of detection; LoQ, limit of quantitation (at 20% coefficient of variation).

| Method comparison
A method comparison study between LiCA and Cobas was performed using a larger pool of serum specimens across the analytical measuring range in our lab. Figure 1 Table 5 as a quick overview.

| Clinical performance
ROC curves were plotted in Figure 2 to describe the diagnostic accuracy of the assays. Using the five-assay panel (TSH, FT3, and FT4) revealed a comparable diagnostic accuracy for hyperthyroidism ( Figure 2B) and hypothyroidism ( Figure 2D) between LiCA and DxI, Cobas, or Centaur, while the AUC was slightly lower than that generated by the five-assay panel.

| DISCUSS ION
The essential principle of LiCA technology is singlet oxygen channeling through two kinds of nanoscale latex beads-sensitizer and emission. 7 In conclusion, LiCA 800 provides a precise and high-throughput immunoassay platform for the detection of thyroid hormones. It is acceptable for clinical use.

CO N FLI C T O F I NTE R E S T
All authors state no conflict of interests.

PATI E NT CO N S E NT
All samples used in this study were collected from residual and deidentified patient sera that were clinically tested for the thyroid assays. A waiver of the informed consent has been approved by the Biomedical Ethics Committee of Peking University International Hospital (No. 2021-KY-0002-01).

PE R M I SS I O N TO R E PRO D U CE M ATE R I A L FRO M OTH E R S O U RCE S
Not applicable.

CLI N I C A L TR I A L R EG I S TR ATI O N
Not applicable.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets generated during the current study are available from the corresponding author on reasonable request.