Real-Time Viability Assessment During Normothermic Machine Perfusion With Resonance Raman Spectroscopy

Background: Viability assessment during normothermic machine perfusion (NMP) would hugely benefit from novel and objective metrics that aid fast and efficient clinical decision making during NMP in order to minimize discard while optimizing transplant outcomes. Real-time perfusate analysis with fluorescent spectroscopy has recently shown to accurately predict post-transplant outcomes during hypothermic machine perfusion. However, presence of highly fluorescent hemoglobin (both free and cellular) intrinsically limits such promising optical approaches for viability assessment during NMP. Methods: To overcome this limitation, we used Raman spectroscopy and analyzed the blood based perfusate of 6 normothermically perfused human donor livers that were rejected for transplantation. The raw perfusate raman spectrum was measured after 1, 3, and 6 hours of perfusion, using a 441nm excitation wavelength. Separately measured spectra of all perfusate components - including hemoglobin – were subtracted from the raw spectrum using a spectral possessing algorithm. The residual Raman spectrum was compared to other viability parameter during NMP such as lactate, resistance, transaminases, bile production and quality. Results: A clear residual spectrum was obtained from the perfusate with distinct peaks at ~1267, ~1505, and ~1620 cm-1. The intensity of this spectrum significantly increased during perfusion for all livers. 4 out of 6 livers met the transplantable viability criteria that are used in European clinical trials. These livers had significantly lower peak heights at all time point compared to the 2 lives that performed poorly during NMP (P=0.030, P=0.006 and P=0.002 at T=1, T=3 and T=6 hours). Conclusion: Raman spectroscopy enables optical analysis of blood based perfusates. This may provide fast and objective viability assessment during NMP in efforts to alleviate the donor organ shortage. National Institute of Health.