An evaluation of the performance of the Point of Care Test iCHROMATM AFP method: Precision and accuracy

(page number not for citation purpose) Journal of Global Medicine 2021. © 2021 J Bolodeoku et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/), allowing third parties to copy and redistribute the material in any medium or format and to remix, transform, and build upon the material for any purpose, even commercially, provided the original work is properly cited and states its license. Citation: Journal of Global Medicine 2021, 1: 7 http://dx.doi.org/10.51496/jogm.v1.7 SHORT REPORT An evaluation of the performance of the Point of Care Test iCHROMATM AFP method: Precision and accuracy

A lpha-fetoprotein (AFP) is a carcinoembryonic glycoprotein present in abundance in foetal blood, with <15 ng/mL in normal adults. Its measurement can significantly increase the specificity at lower levels (i.e. between 10 and 500 ng/mL). These are available but have, to date, been too complex to be widely applied in clinical practice. Serum AFP estimation is useful in the diagnosis and monitoring of primary hepatocellular carcinoma, hepatoblastoma, non-seminomatous testicular germ cell tumours and other germ cell tumours. Estimation of AFP levels in maternal serum and amniotic fluid have been a useful tool in the pre-natal diagnosis of many foetal disorders, such as Down's syndrome, spina bifida, anencephaly and other neural tube defects [1][2][3][4][5][6]. There are a very few points of care test (POCT) methods for measuring AFP that have data in their literature. One of such methods shows the performance of a rapid quantitative method with a gold immunochromatographic strip, which showed a good correlation (r 2 = 0.961) with a chemiluminescent immunoassay analyser [7]. We have extensively evaluated the comparative performance of the qualitative point of care device Boditech iCHROMA™ for estimating prostate specific antigen (PSA) [8][9][10], vitamin D [11], human chorionic gonadotrophin (HCG) [12], luteinizing hormone (LH) [12], follicle stimulating hormone (FSH) [12], C-reactive protein (CRP) [13], microalbumin [13] and TSH [14], and found a very good correlation with other traditional laboratory methods. In this study, we set out to evaluate the precision and accuracy performance of the Boditech iCHROMA™ AFP method, a rapid POCT method that uses a very small amount of blood and can be performed on whole blood, with an analysis time of 15 min, and compared its performance with the traditional laboratory AFP methods enrolled in the Randox International Quality Assessment Scheme (RIQAS).

Materials and methods
The Boditech iCHROMA™ uses a sandwich immunodetection principle, such that the fluorescence-labelled detector antibody binds to the target protein in the sample. The sample is then applied onto a test strip and the fluorescence labelled antigen-antibody complex is captured by a second antibody embedded in the solid phase. The signal intensity of fluorescence of the captured complex is directly proportional to the amount of AFP present and thus allows for the calculation of sample AFP concentration and the result is displayed on the reader as nanograms per millilitre (ng/mL). A fluorescence-labelled control protein is included in the reaction and the intensity of the control line is measured as a quality check.

AFP concentration method
The assay was performed following the manufacturer's instructions:

Part 1
Precision tests the ability of the tests to be repeated on the same device; there are two types of precision tests, withinand between-run tests. In this study, the within-run precision was estimated using the universal control provided by the manufacturer. The control was made up with water and run 25 times on the iCHROMA™ using the AFP method described previously using 15 μL. The mean, standard deviation (SD) and coefficient of variation percent (CV%) were then estimated from the data. The coefficient of variation (CV) is defined as the ratio of the standard deviation to the mean. It is used to express the precision and repeatability of an assay.

Part 2
The accuracy study was carried out with external quality control material (EQA) samples 1-12 of cycles 40 purchased from the RIQAS. These samples (1-12) were constituted and run-in duplicate on the Boditech iCHROMA™ using the method described. The results were compared with the mean results of the following laboratory methods that were registered with the RIQAS database: Abbott Architect, BioMerieux VIDAS, Roche Cobas 6000/8000, Siemens Centaur XP/XPT/Classic, Siemens/DPC/Immulite 2000/2500, Siemens/DPC/ Immulite 1000, Beckman Dxl 600/800, Roche Elecsys, Roche Cobas 4000/e411, Roche Modular E170, SNIBE Maglumi and Ortho Vitros 3600/5600/ECi. The results were plotted in a spreadsheet using an XY plot. The iCHROMA AFP method was plotted on the Y axis and the other laboratory methods on the X axis. A linear regression line was inserted through the data points, and the slope and Y intercept were calculated. The best-fit line will be defined by the equation: Y =m + b, where m is the slope and b is the intercept. The degree of association was measured by a correlation coefficient (R 2 ) on a scale that varies from +1 through 0 to -1.

Part 1 Precision
Twenty-four (96%) of the 25 tests had values that fell within the range of the universal control (lower limit 16.25 ng/mL, an average 21.67 ng/mL, an upper limit 27.09 ng/mL), except for the fourth test with a value of 34.73 ng/mL, see Fig. 1. The Boditech iCHROMA™ AFP intra-assay results ranged from 22.25 to 34.73 ng/mL, with a mean value of 24.5 ng/mL, a SD of ±2.4 ng/mL and a coefficient of variation percent (CV%) of 9.8%.

Part 2
The mean values of the duplicate iCHROMA™ AFP results of the RIQAS samples, 1-12, were 21. 10

Correlations
The coefficient of correlation measures the strength of a possible linear relationship between the other methods. The plots in Fig. 2A-L show the results of the various method estimation comparisons with the 12 quality control samples analysed using the iCHROMA™ AFP method. The coefficient of correlations ranged between + 0.9457 and + 0.9738.

Discussion
Most AFP measuring immunoassays have a high sensitivity and specificity but could be time consuming, expensive and complex. The value of a POCT to measure AFP is, therefore, invaluable. A simple and rapid point-of-care system that uses Eu (III) chelate microparticles with lateral flow immunoassay (LFIA) had been described to determine AFP in serum with an assay time of 15 min and excellent correlation (r = 0.9860) [15]. In this study, another POCT using LFIA, the iCHROMA™ AFP method showed a very good within-run precision of 9.8%, showing that the repeatability of the test performance is good with 95% of the estimated values falling within two standard deviations of the mean (19.7-29.3 ng/mL). There was a consistent overestimation with the iCHROMA™ AFP method compared with the other laboratory methods. The laboratory method's percent bias to the the iCHROMA™ AFP method ranged from 38% in Roche Cobas 6000/8000 and Roche Elecsys to 53% in BioMerieux Vidas. The difference (positive bias) observed between the values of the iCHROMA™ AFP method and the other traditional laboratory methods was consistent and could be due to calibration or the set point on the iCHROMA™ AFP method. Despite the overestimations of the iCHROMA™ AFP method, there was a very good correlation with the methods ranging from r 2 = 0.9457 with SNIBE Maglumi to r 2 = 0.9738 with Roche Cobas 6000/8000 (Fig. 2). This confirms that the iCHROMA™ AFP method's results compare very well with those of the other traditional laboratory methods of measuring AFP, and the constant positive bias observed is probably as a result of a calibration or set point of the iCHROMA™ AFP method, thus suggesting that the reference range of the iCHROMA™ AFP method should be set up taking into consideration a proportional error of 38-52.
In summary, the iCHROMA™ AFP has excellent correlations with traditional laboratory methods, and the assay has a within-run precision value of <10%. These data here show that the iCHROMA™ is a very practical solution for laboratories that require to assay for AFP at the point of care.