Transforming kidney transplant monitoring with urine CXCL9 and CXCL10: practical clinical implementation

Abstract

In kidney transplant recipients, urine CXCL9 and CXCL10 (uCXCL9/10) chemokines have reached a sufficiently high level of evidence to be recommended by the European Society of Organ Transplantation for the monitoring of immune quiescence. To assess the risk of acute rejection (AR), the advantage of uCXCL9/10 is their cost-effectiveness and their high diagnostic performance. Here, we evaluated the feasibility of a next-generation immunoassay for quantifying uCXCL9/10 levels. It demonstrated high efficiency with minimal workflow and a 90-min time to result. Preanalytical studies indicated stability of uCXCL9/10 levels and analytical studies confirmed excellent linearity and precision. In a cohort of 1048 samples collected at biopsy, the results correlated significantly with ELISA quantification and were integrated into a previously validated 8-parameter urine chemokine model. The next generation immunoassay achieved an accuracy of 0.84 for AR diagnosis. This study validates this technology as a robust, locally available and unexpensive platform and marks a significant step towards the widespread implementation of uCXCL9/10, for immune quiescence monitoring. Therefore, we developed an open-access web application using uCXCL9/10 to calculate AR risk and improve clinical decision-making to perform biopsy, ushering in a new era in kidney transplantation, where personalized, data-driven care becomes the norm.

Figure 1

Overview on clinical workflow steps and research validation studies performed to assess Ella® quantification of urine chemokines: bringing research innovation to daily care. Left panel illustrates the 4-step workflow if urine chemokine were used in daily practice: sample collection and storage, assay preparation, chemokines quantification and acute rejection risk assessment. For each clinical step, the middle panel shows the corresponding validation studies which were conducted, independently from manufacturer’s certification. The right panel presents the web application (https://wise-master.admin.semeia.io/bioptim_computation) that was developed to prompt the use of urine chemokines in daily practice. A screen shot of the user interface is shown, illustrating how clinical and biological data can easily be filled in. Abbreviations: AUC, area under the curve; CV, coefficient of variation; LOQ, limit of quantification; min, minutes; prep, preparation; PI, protease inhibitors; RT, room temperature.

Figure 2

Effects of preanalytical sample processing on urine chemokine assessment. (a) Sample preparation study. Correlation plots illustrating relationships between uCXCL9 (left panel) and uCXCL10 (right panel) measurement from 25 pairs of urine samples, prepared and stored with or without protease inhibitors. P-value and r from a Spearman correlation test. (b, c). Sample storage study. Changes in urinary chemokine concentration over various processing delay (24H, 48H and 72H) and at 4 °C (Panel b) or room temperature (Panel c) storage conditions. Raw data from 5 individual samples are presented as solid lines for CXCL9 (navy blue dots) and for CXCL10 (green triangles). The percentage change was calculated at each timepoint in comparison to reference sample, and mean percentage change at each timepoint is presented by dashed lines (navy blue for CXCL9 and green for CXCL10). RM-ANOVA was performed to assess intra-patient variability. CV was calculated between all 4 urinary chemokine levels and mean CV is given as the mean of all intra-patient CVs. Baseline refers to standard sample processing (within 3H after collection) and storage (freezing at − 80 °C).

Figure 3

In-house validation of Ella® assay performance for chemokine quantification from a urine sample. (a, b). Linearity study. (a) Histograms showing uCXCL9 (upper) and uCXCL10 (lower) levels from 10 KTRs urine samples, serially diluted (1:2, 1:4, 1:8 and 1:16) before quantification. For each patient, CV was calculated between each diluted sample and the reference sample (1:2 dilution), and mean CV is given as the mean of all intra-patient CVs. (b) Correlogram illustrating correlation between uCXCL9 (upper) and uCXCL10 (lower) levels after serial dilution for the same 10 KTRs. R values from a Spearman correlation, all P-values < 0.0001. (c, d) ELISA to Ella® correlation study. (c) Correlations between uCXCL9 (N = 1048, navy blue dots) and uCXCL10 (N = 1048, green triangles) levels when measured by ELISA (x-axis) or by Ella® (y-axis) methods. R values from a Spearman correlation, all P-values < 0.0001. (d) Bland-Altman analysis. For each chemokine, Ella®/ELISA ratio (y-axis) is plotting against average chemokine levels (x-axis). Ella®/ELISA ratio of 1 would indicate perfectly superimposable values resulting from both techniques. Bias refers to deviation from this ideal ratio. For representation purpose, only average values < 500 pg/mL are shown. (e) Diagnostic accuracy (C-statistics) of Ella® results tested against reference ELISA results. ROC curves illustrating the diagnostic performance of the 8-parameter chemokine model for any acute rejection, when trained on Ella® or ELISA results (N = 976 samples with no missing value).

Figure 4

Building of a web application for clinicians. An open access web application was developed to prompt the use of urine chemokines in daily practice (https://wise-master.admin.semeia.io/bioptim_computation). (a) The web application offers a simple interactive interface. Users may now easily enter their patients’ clinical data (age, gender), serum lab tests (creatinine, DSA and BKV viral load) and urine lab tests (creatinine, uCXCL9 and uCXCL10 levels), and rapidly run the risk score. Example for a 40-year old woman, with a urine chemokine assessment performed in March 2024. She presents with a 55 mL/min eGFR, has a history of DSA but no current blood BK virus replication or UTI. (b) A screen shot of the results interface is shown, computing an individualized risk of rejection according to the 8-parameter model including Ella®-measured urine chemokines. Using the same clinical and blood biological data as in Panel a, 3 distinct urine chemokine patterns were run in the application and their respective acute rejection risk score is depicted, illustrating the additive value of uCXCL9 and uCXCL10 in a DSA positive KTR.