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Background: Periprosthetic joint infection (PJI) is one of the most serious complications following total knee arthroplasty (TKA). However, the diagnosis remains a challenge for clinicians. In 2011, the muscoskeletal infection society (MSIS) criteria provided a consensus which has been updated in 2013, but these criteria are complex and contain tests that are time-consuming. The same is applicable to the pro-Implant guidelines. Therefore, a simpler diagnostic test is desirable., Objectives: The value of neutrophil gelatinase-associated lipocalin (NGAL), leucocyte esterase (LE) levels, and the white blood cell (WBC) count in synovial fluid to diagnose PJI after TKA was evaluated., Methods: In a retrospective cohort study, we analyzed 89 synovial fluid samples from 86 patients with suspected PJI after TKA. Thirteen and 23 of those samples were classified as PJI according to the MSIS and pro-Implant criteria, respectively. Subsequently, NGAL, LE levels, and the WBC count were determined, the former one using an immunoassay. Using either the MSIS or pro-Implant criteria as the golden standard for PJI, sensitivity and specificity of those markers were determined with ROC curves, and medians were compared with Mann-Whitney U and Pearson Chi-square tests., Results: When applying the MSIS criteria, NGAL revealed 92% sensitivity and 83% specificity. WBC count showed similar sensitivity (92%) and specificity (84%), whereas sensitivity and specificity for LE were 39% and 88% respectively. When applying the pro-Implant criteria, sensitivity was 95% and specificity was 95% for NGAL. Sensitivity and specificity for WBC count were 100% and 97% and for LE 39% and 92% respectively., Conclusion: NGAL and WBC count in synovial fluid has high accuracy in the diagnosis of PJI after TKA and should seriously be considered as part of PJI diagnostics. Leucocyte esterase can serve as rule-in criterion peroperatively. These conclusions are independent of which criteria set was used as golden standard.