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Near-infrared Indocyanine Green Florescence As A Viability Measure During Ex Vivo Normothermic Limb Perfusion
Robert Craig Clark, BS, Varun Kopparthy, PhD, Payam Sadeghi, MD, Abigail Meyers, BS, Sonia Pandey, MD, Ali Abbaszadeh-Kasbi, MD, Frank Papay, MD, Antonio Rampazzo, MD, Bahar Bassiri Gharb, MD, PhD.
Cleveland Clinic Department of Plastic Surgery, Cleveland, OH, USA.

PURPOSE:
Ex vivo normothermic limb perfusion (EVNLP) is a practicable method for preservation of amputated limbs described in animal models to be a feasible alternative to static cold storage for up to 24 hours. Design of a system for prolonged limb preservation necessitates development of endpoint criteria to aid clinical judgement in determination of limb viability. Multifactorial post-perfusion viability assessment of EVNLP limbs is not established. This study correlates quantitative indocyanine green (ICG) florescence data with demonstrated viability measures.
METHODS:
Nine Yorkshire pig forelimbs were preserved by EVNLP utilizing a colloid perfusate with oxygenated HBOC-201 carrier for up to 24 hours. At perfusion endpoint, ICG (7.5mg/3ml) was administered over 20s by arterial line and florescence recorded utilizing the Stryker SPY-Elite System. Between 20s and 60s after ICG induction, six static limb images of foot (n=3) and forelimb (n=3) were cropped from each recording. Image median pixel brightness was determined utilizing ImageJ software and normalized to account for variation in recording conditions. Normalized florescence outputs were compared with weight gain, extensor and flexor compartment pressures, and change in vascular resistance.
RESULTS:
EVNLP continued 213 hours with endpoint weight gain of 22.217%, extensor and flexor compartment pressures of 51.134.2mmHg and 51.131.9mmHg respectively, and increase in vascular resistance of 49.537.3%. Initial limb weight significantly correlated with normalized limb florescence [Forelimb: (r=0.728, p=0.03), Foot: (r=0.734, p=0.02)]. Flexor compartment pressure significantly correlated with extensor compartment pressure (r=0.906, p<.001) and weight gain (r = 0.671, p=0.05).
Normalized limb florescence significantly correlated with weight gain [Forelimb: (r=-0.769, p=0.02), foot (r=-0.801, p=0.01)] but did not correlate significantly with other variables. When controlling for initial limb weight, normalized limb florescence correlated significantly with weight gain [forelimb: (r=-0.823, p=0.01), foot: (r=-0.786, p=0.01)], and normalized foot florescence correlated significantly with flexor compartment pressure (r=-0.692, p=0.04).
CONCLUSION:
Decreasing endpoint limb florescence significantly correlated with increasing weight gain and compartment pressure. Weight gain was previously shown to be a reliable marker of histologically and biochemically verified muscle injury and increased compartment pressure is associated with poor clinical outcomes. This indicates that decreased ICG florescence could be an indicator of limb injury during EVNLP. Further study should include a development of an invariant standardized system for ICG florescence recording and quantification, intra-EVNLP ICG florescence analysis, and application to other grafts.


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