Table 1: Validation parameters.

Validation Parameters			Definition and Criteria
I	Specificity	Ability to detect and differentiate biomarker from other (related) substances
II	matrix effects	Interfering sample matrix component(s) may not affect accuracy
III	Accuracy	Measured and nominal biomarker concentration differ ≤ 15%
IV	Dilution linearity^*	Extra dilution of quality controls exceeding ULOQ is accurate and precise
V	Dilution integrity^#	Normal dilution of samples is accurate and precise
VI	Sensitivity/LOD	Lowest biomarker concentration that is significantly above background level
VII	LLOQ	Lowest concentration that can be measured with accuracy and precision ≤ 20%
VIII	Precision	Variation in concentration between repeated measurements ≤ 15%
IX	Quality controls	Prepared in matrix independently from standards; run together with samples to assure validity; should be within 20% of their nominal concentration
X	Storage stability	Effect storage conditions on biomarker concentration ≤ 15%

Definition and main criteria of the biomarker validation parameters from the Food and Drug Administration (FDA) Bioanalytical Method Validation (BMV) guideline 2018 (5). * Because of the narrow range of the calibration standard curve, it is necessary to demonstrate with quality control samples that the analyte of interest, when present in concentrations exceeding the range of quantification (above ULOQ), can be accurately measured by the assay after dilution in blank matrix to bring the biomarker concentrations into the validated range for analysis. An additional reason for conducting dilution linearity experiments is to detect a possible prozone or “hook effect” for ligand binding assays. # The effect of dilutions used for samples with normal biomarker concentrations on accuracy and precision. LOD = limit of detection; ULOQ and LLOQ = upper and lower limit of quantification.