ABSTRACT
[Abstract] Objective To analyze the difference of the carbohydrate antigen 242 (CA242) results measured by Luminex flow cytometry fluorescence immunoassay with different calibrations and different ambient temperatures, so as to evaluate the effect of ambient temperature on the measurement results. Methods Two CA242 samples with different concentrations (median- and high-value) were selected and the determination was repeated at 11 temperature points (18.3, 20.1, 21.5, 22.2, 24.1, 25.0, 25.4, 26.5, 27.4, 28.5, and 30.0 ℃) for 5 times by Luminex flow cytometry fluorescence immunoassay. The average value of the detection result at the calibration temperature (25 ℃) was taken as the reference value, and the biases of the detection results at different ambient temperatures were calculated. Forty-nine CA242 specimens covering high-, median- and low-values were examined at 20, 25 and 30 ℃ by Luminex flow cytometry fluorescence immunoassay, and the results were compared and correlation analysis was carried out. Results The biases of the median- and high-value samples at 18.3 ℃ were －35.6% and －29.4%, respectively. The difference between the measurement results at 20 ℃ and 25 ℃ was significant for the 49 specimens (P0.001), and there was a linear correlation between them (Y＝0.676 9X＋0.374 7, R2＝0.990 5). The difference of measurement results was significant between 30 ℃ and 25 ℃ (P0.001), and there was a linear correlation between them (Y＝0.896 6X＋0.227 0, R2＝0.999 4). Conclusion Too low or too high ambient temperature has a negative effect on the determination of CA242 by Luminex flow cytometry fluorescence immunoassay. The ambient temperature should be kept relatively constant and controlled within the range of calibration temperature ±4 ℃.
ABSTRACT
[Abstract] Objective To establish a new method for detecting Mycobacterium tuberculosis (TB) infection based on flow cytometry fluorescence immunoassay and interferon γ release assay (IGRA). Methods The whole blood samples were stimulated to produce interferon γ (IFN-γ) with phytagglutinin (PHA) and TB specific mixed peptides (early secretory antigenic target [ESAT-6] and culture filtrate protein 10 [CFP-10]), and the plasma was analyzed by double antibody sandwich method combined with flow cytometry fluorescence immunoassay. The IFN-γ concentration was evaluated by receiver operating characteristic (ROC) curve for the diagnostic efficacy of TB. The linear range, minimum detection limit, repeatability, anti-interference performance of the established method were observed, and the consistency of detection with similar products on the market was evaluated. Results The linearity of the flow cytometry fluorescence immunoassay ranged from 2 pg/mL to 1 000 pg/mL. The lowest detection limit was 0.3 pg/mL; the repeatability parameters (coefficient of variation) of the samples at 100 pg/mL and 500 pg/mL were 4.58% and 2.46%, respectively. The average recovery rate of recovery assay was 98.0%. There was no interference with flow cytometry fluorescence immunoassay when the highest concentrations of triglyceride, bilirubin and hemoglobin were 50 mg/mL, 0.6 mg/mL and 10 mg/mL, respectively. As the optimum cut-off value of the IFN-γ concontration was 10 pg/mL, the sensitivity of IFN-γ in diagnosis of TB infection was 82.46% and the specificity was 87.30%. The total coincidence rates with T-SPOT, QFT, and Wantai TB-IGRA reagent were 97.2%, 83.0%, and 85.4%, respectively; and the Kappa coefficients were 0.822, 0.622 and 0.630, respectively. Conclusion The method for diagnosis of TB infection established in this study has a good performance, with the accuracy reaching the level of similar products on the market, and our method has obvious advantages in terms of repeatability and detection process.