Early risk prognosis of free-flap transplant failure by quantitation of the macrophage colony-stimulating factor in patient plasma using 2-dimensional liquid-chromatography multiple reaction monitoring-mass spectrometry

Abstract

Although great success of microvascular free-flap transplantation surgery has been achieved in recent years, between 1.5% and 15% of flaps are still lost due to vascular occlusion. The clinical challenge remains to salvage a transplant in the case of vascular complications. Since flap loss is devastating for the patient, it is of utmost importance to detect signs of complications or of conspicuities as soon as possible. Rescue success rates highly depend on early revision. In this study, we collected blood samples during transplantation surgery from either the contributory artery or the effluent vein of the flap and applied a targeted mass spectrometry-based approach to quantify 24 acute phase proteins, cytokines, and growth factors in 63 plasma samples from 21 hospitalized patients, generating a dataset with 9450 protein concentration values. Biostatistical analyses of the targeted plasma protein concentrations in all 63 plasma samples showed that venous concentrations of macrophage colony-stimulating factor (M-CSF) provided the highest accuracy for discriminating patients with either clinical conspicuities or complications from control individuals. Using 21.33 ng/mL of M-CSF as the diagnostic threshold when analyzing venous blood plasma samples, the assay obtained a sensitivity of 0.93 and a specificity of 0.85 with an area under the curve value of 0.902 in the receiver operating characteristic analysis. Overall, our results indicate that M-CSF is a potential molecular marker for early risk prognosis of free-flap transplant failure.

Figure 1

Exemplary cases for free tissue transfer. (A) Patient after amputation of the left breast. (B) Same patient 6 months after the 1st step of microvascular breast reconstruction with a transplant of skin and fatty tissue from the belly (deep inferior epigastric artery perforator [DIEP] flap). The green spot marked on the left breast represents the position of the later nipple-areola complex that will be reconstructed in the 2nd and last step of breast reconstruction. (C) Another patient after DIEP flap transplantation. In the area marked with (I) the flap develops good, in region (II) there is a lack of perfusion. There is an intermediate zone in between (green).

Figure 2

ROC analysis of plasma M-CSF concentrations from vein 2 samples. Calculated AUC is 0.908. The vertical solid line indicates the maximum Youden index (J max), the hatched diagonal indicates the 0.5 value. AUC = area under the curve, M-CSF = macrophage colony-stimulating factor, ROC = receiver operating characteristic.

Figure 3

Logistic regression analysis of plasma M-CSF concentrations from the vein 2 group. COCO (N = 7) and CTRL (N = 14). Outliers are labeled with patient ID numbers. CTRL = control, M-CSF = macrophage colony-stimulating factor.

Figure 4

Logistic regression analysis of plasma M-CSF concentrations from the combined vein 1 plus vein 2 groups. COCO (N = 14) and CTRL (N = 27). Outliers are labeled with patient ID numbers. CTRL = control, M-CSF = macrophage colony-stimulating factor.

Figure 5

ROC analysis of plasma M-CSF concentrations from combined vein 1 plus vein 2 samples. Calculated AUC is 0.902. The vertical solid line indicates the maximum Youden index (J max), the hatched diagonal indicates the 0.5 value. AUC = area under the curve, M-CSF = macrophage colony-stimulating factor, ROC = receiver operating characteristic.

Figure 6

Logistic regression analysis of plasma M-CSF concentrations. Plasma samples from the combined vein 1 plus vein 2 groups. COCO (N = 14) and CTRL (N = 27). Samples that fall within the safety margins (shaded area; 19.3–23.4 ng/mL) are excluded. CTRL = control, M-CSF = macrophage colony-stimulating factor.