*3.2. Methods*

Blood samples were drawn from peripheral veins after an overnight fast. Samples were clotted for 30 min, centrifuged at room temperature, 1000 g for 15 min, then serum was stored at −80 ◦C until assayed. Urine was collected aseptically from the first morning sample, centrifuged at room temperature, 1000 g for 15 min and then stored at −80 ◦C until assayed.

The serum and urine concentrations of clusterin, cystatin C and KIM-1 were evaluated by commercially available ELISA kits (clusterin EIAab, reagen<sup>t</sup> kit E1180h; cystatin C R & D Systems, reagen<sup>t</sup> kit DSCTC0; KIM-1 EIAab, reagen<sup>t</sup> kit E0785 h). Standards, serum and urine samples were transferred to 96-well microplates precoated with recombinant antibodies to human clusterin, cystatin C, KIM-1 and creatinine. Captured proteins were then detected using monoclonal antibodies against clusterin, cystatin C and KIM-1 conjugated to horseradish peroxidase. Next, the assay was developed with tetramethylbenzidine substrate and blue color was developed proportionately to the amount of captured protein. The addition of acid stop solution ended the color development and converted it to the endpoint yellow. The intensity of the latter was measured in a microplate reader at 450 nm, with the correction wavelength at 550/650 nm. Each sample was tested in duplicate and the arithmetical mean was considered a final result. Measurements were performed according to the manufacturer's instructions; results were calculated by reference to standard curves. Detection limits were as follows: clusterin 1.56 ng/mL; cystatin C 3.13 ng/mL; KIM-1 0.15 ng/mL. The intra-assay and inter-assay coefficients of variation (% CV) for examined parameters did not exceed 8.5% and 9.4%, respectively.

The assessment of kidney function relied on hematological protocols assessing serum creatinine in fixed time points. Serum and urine chemistry parameters were measured using automated routine diagnostic tests on the Beckman Coulter AU2700 analyzer. The serum creatinine was assessed with the use of enzymatic method (creatinine OSR61204 reagent, creatininase–sarcosine oxidase reactions). Serum and urine concentrations of all parameters was measured before conditioning, 24 h after allotransplantation and then 1 week, 2, 3, 4 weeks after alloHSCT. eGFR was calculated in all time points, based on the Schwarz formula [18]. The eGFR changes were confronted with the pre-transplantation values.

All urinary concentrations of evaluated parameters were normalized to urinary creatinine values. AKIwasdiagnosedbasedonthepRIFLEcriteria [9].HyperfiltrationwasdefinedaseGFR ≥ 140

mL/min/1.73 m2, according to recent meta-analysis and pediatric experience [19,20].

## *3.3. Statistical Analysis*

Results were expressed as median values and interquartile ranges. The null hypothesis of normality of distribution of analyzed variables was rejected by Shapiro–Wilk test. Thus, the comparisons between paired and unpaired data were evaluated by using nonparametric tests (Friedman, Wilcoxon, Kruskal–Wallis, Mann–Whitney U). The correlations between parameters were assessed with the use of Spearman's correlation coe fficient R. Statistical analysis was performed using the package Statistica ver. 13.3 (StatSoft). A *p*-value < 0.05 was considered significant.
