Synovial Fluid Leukocyte Count and Differential Are Poor Standalone Rule-in Tests for Periprosthetic Joint Infection: Results of a Methodological Audit and Reanalysis of the 2025 Meta-analysis Underpinning the Unified Periprosthetic Joint Infection Criteria.

Abstract

The Unified Periprosthetic Joint Infection (PJI) Criteria were developed and endorsed by the European Society of Clinical Microbiology and Infectious Diseases, the European Bone and Joint Infection Society, the Infectious Diseases Society of America, and the Musculoskeletal Infection Society and formally presented at the 2025 International Consensus Meeting. These criteria recommended that synovial fluid white blood cells (WBC) > 3000 cells/µL or polymorphonuclear cells (PMN) > 75% each should be considered individually sufficient to confirm a diagnosis of PJI as standalone rule-in tests, stating specificity of > 95%. These thresholds and performance align with a 2025 meta-analysis published on behalf of the Unified PJI Definition Task Force, reporting specificities of 98.5% and 97.8%, respectively. Because such near-perfect specificities conflict with prior meta-analyses and known causes of false-positive leukocyte testing, independent validation of the supporting evidence is warranted. (1) Is the 2025 meta-analysis published on behalf of the Unified PJI Definition Task Force methodologically rigorous and reproducible under independent appraisal and audit? (2) Do WBC count and PMN percentage demonstrate sufficient and generalizable rule-in performance for PJI? An independent consortium with expertise in PJI diagnostics, epidemiology, and biostatistics conducted a formal methodological reappraisal of the 2025 meta-analysis. To address the first study question, the 2025 meta-analysis, all 74 primary studies, and the original source data files provided by that study's authors were assessed. After reproducing the original results of the 2025 meta-analysis to confirm understanding of data files and workflow, two independent reviewers used A Measurement Tool to Assess Systematic Reviews (AMSTAR-2) to assess quality, and then a methodological audit was performed using the Cochrane Handbook for Systematic Reviews of Diagnostic Test Accuracy. Limitations were graded by consensus as critical, major, or minor, with critical limitations independently verified by three statisticians and two clinicians. To address our second study question, a reanalysis of the same corpus of 74 studies was performed using a hierarchical bivariate random-effects model, restricted to studies with predetermined thresholds. A standalone rule-in test is one in which a positive result alone is sufficient to establish the diagnosis of PJI, and its performance is most appropriately assessed using positive predictive value (PPV), which directly reflects the probability that a positive result represents true PJI. Sufficient rule-in performance was pragmatically defined as a PPV of > 90% across clinically plausible prevalence rates. Sufficiently generalizable performance was defined as demonstrable consistency across institutions, assessed through between-study heterogeneity of specificity. For the first study question, AMSTAR-2 assessment rated the 2025 meta-analysis as critically low overall confidence, reflecting weaknesses in multiple domains. Three critical limitations were identified during audit: (1) a data integrity discrepancy in the software input table used for the 2025 meta-analysis, in which false-positive counts were placed in the false-negative column and false-negative counts were placed in the false-positive column for all studies, rendering downstream results unreliable; (2) an outcome-dependent (self-fulfilling) threshold-selection approach that included only studies already reporting > 95% sensitivity or specificity, yielding results that were mathematically constrained to meet the 95% performance criteria, followed by derivation of thresholds using unweighted medians rather than meta-analytic techniques; and (3) presentation of pooled estimates and clinical threshold recommendations despite acknowledgment of markedly high heterogeneity (I2 statistic 88.3% to 99.3%). Major limitations included unit-of-analysis errors, use of specificity alone (rather than PPV) to define rule-in performance, unaddressed incorporation bias, and reliance on data-driven (Youden index) threshold selection. Regarding our second study question, in a reanalysis of the same 74 source studies, we found that WBC count and PMN percentage did not demonstrate sufficient or generalizable rule-in performance for PJI. Specifically, the heterogeneity for specificity remained high (I2 = 85% for WBC and I2 = 93% for PMN), reflecting considerable between-study variability. Summary specificity was 90% (95% confidence interval [CI] 86% to 92%) for WBC and 92% (95% CI 87% to 96%) for PMN. Resulting PPVs were low across clinically plausible disease prevalence rates, with point estimates of 67% (95% CI 61% to 74%) and 73% (95% CI 63% to 87%), respectively, at 20% PJI prevalence. Critical data integrity discrepancies and methodological limitations substantially undermine the reliability of the 2025 meta-analysis published on behalf of the Unified PJI Definition Task Force. Reanalysis of the same evidence base using recommended meta-analytic methods characterizes WBC count and PMN percentage as poor standalone rule-in tests for PJI for two main reasons: (1) the high heterogeneity of specificity for both WBC count and PMN percentage undermines the generalizability of these tests across institutions and precludes the establishment of universal thresholds, and (2) PPVs at clinically reasonable prevalence rates were low (67% to 73%) for standalone rule-in test usage, meaning that roughly 1 in 3 positive results would likely be a false-positive. We recommend that surgeons not use synovial fluid WBC count or PMN percentage thresholds as standalone rule-in criteria for PJI. Instead, these tests should be interpreted as supportive findings within a multimodal diagnostic framework, particularly when results are discordant with clinical findings, cultures, histology, serum markers, or other synovial tests. Professional organizations and regulatory bodies should reconsider recommending WBC count or PMN percentage as standalone rule-in tests for PJI until methodologically robust evidence demonstrates reproducible thresholds with sufficiently high PPVs.