Neutrophil CD64, CD66b, and Serum Thioredoxin as Early Biomarkers of Delayed Fracture-Related Infection After Internal Fixation: A Prospective Cohort Study

Background: Delayed fracture-related infection (FRI) remains a major complication after internal fixation, and conventional inflammatory markers have limited diagnostic accuracy. This study evaluated neutrophil CD64, CD66b, and serum thioredoxin as adjunctive biomarkers for delayed FRI. Methods: In this prospective cohort study at a tertiary trauma center, 637 patients undergoing internal fixation for long-bone fractures were enrolled and followed until fracture union. Venous blood was collected pre-operatively and on postoperative days 3, 7, and 10. Total leucocyte count (TLC), erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), neutrophil CD64 and CD66b, and serum thioredoxin were measured. FRI was defined using contemporary clinicoradiological and microbiological consensus criteria. Diagnostic performance was assessed by receiver operating characteristic (ROC) analysis. Results: Delayed FRI occurred in 56/637 patients (8.8%). FRI was associated with open fractures (30.4% vs 9.0%) and Tscherne grade ≥2 (78.6% vs 61.1%) and significantly prolonged time to union (21.84 ± 6.92 vs 16.26 ± 3.42 weeks; p < 0.0001). From day 3 onward, TLC, ESR, and CRP were significantly higher in the FRI group; CRP on day 7 showed the best conventional performance (cut-off >20 mg/L; sensitivity 83%, specificity 80%, AUC 0.85). CD64, CD66b, and thioredoxin diverged sharply between groups. CD64 on day 10 (cut-off >3800 median fluorescence intensity (MFI)) yielded a sensitivity of 91%, a specificity of 87%, NPV of 99% (AUC 0.93). CD66b on day 3 (>450 MFI) and thioredoxin on day 7 (>85 ng/mL) showed AUCs of 0.87 and 0.89 with NPVs of 98%. Conclusion: Neutrophil CD64, CD66b, and serum thioredoxin outperform conventional markers for detecting delayed FRI and, in combination, provide a high-negative predictive value panel that may substantially improve early diagnosis and safe exclusion of infection after fracture fixation.