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Hypothermic machine perfusion (HMP) provides preservation superior to cold storage and may allow for organ assessment prior to transplantation. Since flavin mononucleotide (FMN) in perfusate has been proposed as a biomarker of organ quality during HMP of donor livers, the aim of this study was to validate FMN as a biomarker for organ quality in the context of HMP preserved kidneys. Perfusate samples (n = 422) from the paired randomised controlled COPE-COMPARE-trial, comparing HMP with oxygenation (HMPO2) versus standard HMP in kidneys, were used. Fluorescence intensity (FI) was assessed using fluorescence spectroscopy (excitation 450nm; emission 500-600nm) and validated by fluorospectrophotometer and targeted liquid chromatography mass spectrometry (LC-MS/MS). Fluorescence intensity (FI)(ex450;em500-600) increased over time during machine perfusion in both groups (p<0.0001). This increase was similar for both groups (p = 0.83). No correlation, however, was found between FI(ex450;em500-600) and post-transplant outcomes, including day 5 or 7 serum creatinine (p = 0.11; p = 0.16), immediate graft function (p = 0.91), creatinine clearance and biopsy-proven rejection at one year (p = 0.14; p = 0.59). LC-MS/MS validation experiments of samples detected FMN in only one perfusate sample, whilst the majority of samples with the highest fluorescence (n = 37/38, 97.4%) remained negative. In the context of clinical kidney HMP, fluorescence spectroscopy unfortunately appears to be not specific and probably unsuitable for FMN. This study shows that FMN does not classify as a clinically relevant predictive biomarker of kidney graft function after transplantation.

低温机器灌注（Hypothermic Machine Perfusion, HMP）的器官保存效果优于冷保存，且可在移植前完成器官质量评估。由于灌注液中的黄素单核苷酸（Flavin Mononucleotide, FMN）已被提议作为供体肝脏低温机器灌注过程中器官质量的生物标志物，本研究旨在验证FMN作为经HMP保存的肾脏器官质量生物标志物的有效性。

本研究采用配对随机对照COPE-COMPARE试验的灌注液样本（n=422），该试验对比了含氧低温机器灌注（HMPO2）与标准低温机器灌注在肾脏保存中的应用效果。采用荧光光谱法（激发波长450nm；发射波长500~600nm）检测荧光强度（Fluorescence Intensity, FI），并通过荧光分光光度计及靶向液相色谱-串联质谱（LC-MS/MS）进行方法学验证。

两组样本的荧光强度（FI，激发450nm；发射500~600nm）在机器灌注过程中均随时间显著升高（p<0.0001），且两组的升高趋势无统计学差异（p=0.83）。然而，荧光强度与移植后多项转归指标均无相关性，包括术后第5天、第7天血清肌酐水平（p=0.11；p=0.16）、即刻移植物功能（p=0.91）、肌酐清除率以及1年时活检证实的排斥反应（p=0.14；p=0.59）。

LC-MS/MS验证实验仅在1份灌注液样本中检测到FMN，而多数荧光强度较高的样本（n=37/38，97.4%）仍呈阴性结果。

综上，在临床肾脏低温机器灌注场景中，荧光光谱法特异性不足，或不适用于FMN的检测。本研究证实，FMN无法作为移植后肾脏移植物功能的临床相关预测生物标志物。