key: cord-0024582-hzii79mo authors: Sun, Chenxi; Wang, Dongxia; Xu, Henggui; Yang, Guang; Yan, Xiaomei; Liu, Hui title: A method for measuring the experimental resolution of laboratory assays (clinical biochemical, blood count, immunological, and qPCR) to evaluate analytical performance date: 2021-11-01 journal: J Clin Lab Anal DOI: 10.1002/jcla.24087 sha: 04afe6affcb766b956806a6d804bb24df0cba5db doc_id: 24582 cord_uid: hzii79mo BACKGROUND: The measurement method for experimental resolution and related data to evaluate analytical performance is poorly explored in clinical research. We established a method to measure the experimental resolution of clinical tests, including biochemical tests, automatic hematology analyzer methods, immunoassays, chemical experiments, and qPCR, to evaluate their analytical performance. METHODS: Serially diluted samples in equal proportions were measured, and correlation analysis was performed between the relative concentration and the measured value. Results were accepted for p ≤ 0.01 of the correlation coefficient. The minimum concentration gradient (eg, 10%) was defined as the experimental resolution. For this method, the smaller the value, the higher the experimental resolution and the better the analytical performance. RESULTS: The experimental resolution of the most common biochemical indices reached 10%, with some even reaching 1%. The results of most counting experiments showed experimental resolution up to 10%, whereas the experimental resolution of the classical chemical assays reached 1%. Unexpectedly, the experimental resolution of more sensitive assays, such as immunoassays was only 25% when using the manual method and 10% for qPCR. CONCLUSION: This study established a method for measuring the experimental resolution of laboratory assays and provides a new index for evaluating the reliability of methods in clinical laboratories. laboratory must verify or establish the method performance specifications that are applicable and clinically relevant". 5 At present, the general performance evaluation indexes include the limit of detection (LoD), accuracy, precision, and linear evaluation. 6, 7 However, there is no relevant index that reflects the minimum measurement difference within a certain concentration range. In many cases, the LoD is used to reflect the sensitivity of a detection system. The LoD refers to the smallest concentration that can be reliably measured by an analytical procedure, which can distinguish 0 from the minimum detection concentration but cannot specify the minimum detectable measurement within a certain concentration range. 8, 9 Reflecting this experimental minimum within the concentration range is an important performance evaluation index, which is related to, yet different from the LoD. However, at present, there is no relevant evaluation index. Therefore, we introduced the concept of "experimental resolution" to address this issue, with the aim of improving the experimental performance evaluation. The experimental resolution is the minimum change that can be detected by an instrument, which should be the basis of the LoD. 10 Thus, the experimental resolution and LoD are related but separate parameters. As the future of medicine is based on effective patient-centered practice, it is therefore important to select test items with appropriate experimental resolution according to the clinical needs. 11 Clinical experiments can be divided into quantitative, semiquantitative, and qualitative assays. 4 The higher the experimental resolution, the better the quantitative effect. Experimental resolution is the key index for evaluating test performance, but no research has been done on a measurement method for experimental resolution or its related data to evaluate test performance. To address these issues, this study adopted a method involving an equal-proportion dilution series of samples and used the improved linear measurement method to measure the experimental resolution of commonly used assays, including clinical biochemical, automatic hematology analyzer, chemical, immunological, and qPCR assays. By analyzing the test results, we found that the experimental resolution of the clinical biochemical experiments and the automatic hematology analyzer experiments were generally higher than 10% but remained lower than traditional chemical experiments (for which the experimental resolution could reach 1%). Surprisingly, the experimental resolution of the immunoassay and real-time fluorescence quantitative assay, which are generally considered to be more sensitive methods, was lower than 1%. [12] [13] [14] By analyzing Pearson's correlation of the correlation coefficients of the results of biochemical samples with different concentration gradients, the results of samples with different dilution ratios could not be predicted, and the experimental resolution should therefore be based on actual measurements rather than relying on a single dilution series. Thus, we propose that the experimental resolution is an important index for the evaluation of experimental performance. For the preparation of samples with a 50% concentration gradient of equal-proportion dilutions, 200 µl normal saline was placed in each Eppendorf (EP) tube, and 200 µl serum was added to the first tube. After thorough mixing, 200 µl diluted sample was taken from the first tube and added to the second tube, which was thoroughly mixed. This was followed by two similar dilutions. Serially diluted samples with relative concentrations of 1000% (undiluted serum), 500%, 250%, 125%, and 62.5% were obtained. For the preparation of samples with a 25% concentration gradient of equal-proportion dilutions, 200 µl normal saline was placed in each EP tube, and 600 µl serum was added to the first tube. After thorough mixing, 600 µl diluted sample was taken from the first tube and added to the second tube, which was thoroughly mixed. This was followed by two similar dilutions. A series of diluted samples with relative concentrations of 1000% (undiluted serum), 750%, 563%, 422%, and 316% were obtained. For the preparation of samples with a 10% concentration gradient of equal-proportion dilutions, 160, 80, 40, and 20 µl normal saline were placed in each of the four EP tubes, respectively, and 1440 µl serum was added to the first tube. After thorough mixing, 720 µl diluted sample was taken from the first tube and added to the second tube, which was thoroughly mixed. Then, 360 µl diluted sample was taken from the second tube and added to the third tube, which was thoroughly mixed. Finally, 180 µl diluted sample was taken from the third tube and added to the fourth tube and thoroughly mixed to obtain a series of diluted samples with relative concentrations of 1000% (undiluted serum), 900%, 810%, 729%, and 656%. For the preparation of samples with a 1% concentration gradient of equal-proportion dilutions, 0.4, 0.2, 0.1, and 0.05 ml normal saline were placed in each of four beakers, respectively, and 39.6 ml serum was accurately measured with an acid burette into the first beaker. After fully mixing, 19.8 ml diluted sample was accurately measured from the first beaker into the second beaker and fully mixed. Then, 9.9 ml diluted sample was taken from the second beaker into the third beaker and thoroughly mixed. Finally, 4.95 ml diluted sample was taken from the third beaker into the fourth beaker and thoroughly mixed to obtain a series of diluted samples with relative concentrations of 1,000% (undiluted serum), 990%, 980%, 970%, and 961%, as shown in Figure 1 . After each serum sample was diluted, albumin (ALB) was mea- Serially diluted positive serum samples of anti-HBV surface antigen (anti-HBs) were detected using an anti-HBs commercial ELISA kit (Shenyang Huimin Biological Technology Co., Ltd) according to the manufacturer's instructions. The HCG test card was used to detect the HCG-positive series of diluted urine samples, and the reaction results were photographed and processed. Image J v1.8.0 was used to process the photographs to obtain the gray scale of the C area of the quality control line and the T area of the test line. The gray scale ratio (T/C) between the test line and quality control line was calculated using the following formula: The concentration of carcinoembryonic antigen in the diluted samples was determined using a Mindray i2000 chemiluminescence analyzer (Shenzhen Mindray Biomedical Electronics Co., Ltd.). The absorbance values of the copper and strontium diluted samples were determined using an SP-3900AA flame atomic absorption spectrometer (Shanghai Spectrum Instruments Co., Ltd). (1) T∕C = the gray scale of the C area the gray scale of the T area and diluted DNA samples were used to construct a qPCR system for the amplification of genes (ie, the 18S ribosomal RNA gene), and the relative DNA concentration N was calculated as follows: where Ct 0 is the Ct value of the undiluted sample and Ct is the Ct value of the treated sample. The linear evaluation method was used to evaluate the experimental resolution. The specific method has been previously reported in the literature, 15 with some modifications: 1. The equal-proportion concentration gradient dilution method was adopted instead of the equal-spacing concentration gradient dilution method for the linear evaluation of the diluted samples. 2. In the original method, the same sample was measured at least twice in parallel. For the purposes of this study, to control the detection range, the same sample was designed to be tested only once. 3. According to the definition of experimental resolution, linear regression was used to analyze the experimental results, with the relative concentration used as the independent variable and the actual test value as the dependent variable for linear fitting. When determining the boundary value, the p-value was reduced from 0.05 to 0.01. Therefore, the modified experimental resolution determination method was as follows: the correlation analysis was conducted between the actual measured values obtained from each exper- The correlation between the detection results of common clinical biochemical indicators of sera with concentration gradients of 25%, 10%, and 1% with the relative concentrations is shown in Table 1 . The results showed a significant p ≤ 0.01 for the correlation test re- The detection results for the experimental resolution of the automatic hematology analyzer are presented in Table S1 . The experi- results of the samples with 25%, 10%, and 1% concentration gradient dilutions, as shown in Table 3 . The experimental resolution refers to the minimum variation that can be detected by an instrument. We believe that the magnitude of the minimum variation can be explained by the merits and demerits of linear fitting. Although imprecision errors or matrix effects can reflect the precision and accuracy of assays, the poor performance of any of the above indicators will affect the experimental resolution. Table 3 ). In addition, for the fitting curve, we only considered whether the fitting result met the requirement of p ≤ 0.01 and did not consider the slope or intercept of the fitting curve. If the fitting effect is good, the accuracy can be further improved by regression. In conclusion, the established determination method for exper- Therefore, the experimental resolution may be considered as a new index for the performance evaluation of clinical trials, which will influence new discoveries resulting from biochemical tests, complete blood count tests, chemical experiments, immunoassays, qPCR, and other medical tests. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this study. Hui Liu and Chenxi Sun designed the experiments. Chenxi Sun and Dongxia Wang performed the experiments. Chenxi Sun analyzed the data and wrote the study. Hui Liu reviewed and edited the study. Henggui Xu, Guang Yang, and Xiaomei Yan provided study materials. All authors have read and approved the final study and take responsibility for its integrity. All relevant data are within the study, and no additional data are available. 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