Added Value of Histological Evaluation of Muscle Biopsies in Porcine Vascularized Composite Allografts

<b>Background</b>: Machine perfusion (MP) offers extended preservation of vascularized complex allografts (VCA), but the diagnostic value of histology using hematoxylin and eosin (H&E) in detecting ischemia-reperfusion injury (IRI) in muscle cells remains unclear. This study aims to document the application of the Histology Injury Severity Score (HISS) and to assess whether additional staining for nicotinamide adenine dinucleotide (NADH) and membrane attack complex (MAC) improves IRI detection in a porcine limb replantation model. <b>Methods</b>: The forelimbs of 16 Dutch Landrace pigs were amputated and preserved for 24 h using hypothermic MP (<i>n</i> = 8) with Histidine-Tryptophan-Ketoglutarate (HTK) or for 4 h with SCS (<i>n</i> = 8) before heterotopic replantation and 7 days of follow-up. Muscle damage was assessed via biochemical markers and light microscopy using H&E, NADH, and MAC at baseline, post-intervention, and post-operative day (POD) 1, 3, and 7 timepoints, using the HISS and a self-developed NADH and MAC score. <b>Results</b>: H&E effectively identified damaged muscle fibers and contributed to IRI assessment in porcine limbs (<i>p</i> < 0.05). The highest HISS was measured on POD 3 between MP (4.9) and SCS (3.5) (<i>p</i> = 0.029). NADH scores of both preservation groups varied over the 7-day follow-up and were statistically insignificant compared with baseline measurements (<i>p</i> > 0.05). MAC revealed no to minimal necrotic tissue across the different timepoints. <b>Conclusions</b>: This study documents the application of the HISS with H&E to detect IRI in muscle fibers. NADH and MAC showed no significant added diagnostic utility. The 24 h MP showed similar muscle alterations using the HISS compared to that of the 4 h SCS after a 7-day follow up.