Synovial fluid alpha-defensin in periprosthetic knee infection workup : liquid chromatography-mass spectrometry detection of alpha-defensin in synovial fluid.

AIMS: The diagnosis of periprosthetic joint infection (PJI) continues to present a significant clinical challenge. New biomarkers have been proposed to support clinical decision-making; among them, synovial fluid alpha-defensin has gained interest. Current research methodology suggests reference methods are needed to establish solid evidence for use of the test. This prospective study aims to evaluate the diagnostic accuracy of high-performance liquid chromatography coupled with the mass spectrometry (LC-MS) method to detect alpha-defensin in synovial fluid. METHODS: Between October 2017 and September 2019, we collected synovial fluid samples from patients scheduled to undergo revision surgery for painful total knee arthroplasty (TKA). The International Consensus Meeting criteria were used to classify 33 PJIs and 92 aseptic joints. LC-MS assay was performed to measure alpha-defensin in synovial fluid of all included patients. Sensitivity, specificity, positive predictive value, negative predictive value, and the area under the receiver operating characteristic curve (AUC) were calculated to define the test diagnostic accuracy. RESULTS: The AUC was 0.99 (95% confidence interval (CI) 0.98 to 1.00). Receiver operating characteristic (ROC) analysis showed that the optimal cut-off value of synovial fluid alpha-defensin was 1.0 µg/l. The sensitivity of alpha-defensin was 100% (95% CI 96 to 100), the specificity was 97% (95% CI 90 to 98), the positive predictive value was 89.2% (95% CI 82 to 94), and negative predictive value was 100% (95% CI 96 to 100). ROC analysis demonstrated an AUC of 0.99 (95% CI 0.98 to 1.0). CONCLUSION: The present study confirms the utility of alpha-defensin in the synovial fluid in patients with painful TKA to select cases of PJI. Since LC-MS is still a time-consuming technology and is available in highly specialized laboratories, further translational research studies are needed to take this evidence into routine procedures and promote a new diagnostic approach.Cite this article: Bone Joint J 2022;104-B(9):1047-1051.

Investigating Adherence to COVID-19 Vaccination and Serum Antibody Concentration among Hospital Workers-The Experience of an Italian Private Hospital.

SARS-CoV-2 transmission has been high, especially among healthcare workers worldwide during the first wave. Vaccination is recognized as the most effective approach to combat the pandemic, but hesitation to get vaccinated represents an obstacle. Another important issue is the duration of protection after administration of the full vaccination cycle. Based on these premises, we conducted a study to evaluate vaccination adherence and the anti-S antibodies levels among hospital workers, from January to March, 2021. To assess adherence, an anonymous questionnaire was used. Anti-S antibody levels were obtained from the monitoring serological sample database. In total, 56.2% of the unvaccinated people did not report a previous infection from COVID-19. Among those who have not been vaccinated, 12.5% showed distrust against the vaccine, 8.3% stated to have received contraindications to the vaccination, and 6.3% did not report any choice. Analyzing anti-S antibody levels, only one person was found to have a value below the lower cut-off, two weeks, and three months after receiving their second dose. One was below the cut-off after two weeks, and then above the same cut-off after three months. The results of our survey should be seen as a stimulus to further sensitize hospital staff to the importance of vaccination and pay attention to anti-S antibody levels monitoring.

Analytical evaluation of direct bicarbonate measurement with the new gem premier chemstat in hemodialysis patients.

GEM Premier ChemSTAT is a whole-blood analyzer designed for providing a rapid basic metabolic panel, inclusive of creatinine and blood urea nitrogen, with the unique characteristic of providing measured bicarbonate (HCO3-) levels. The aim of this work was to evaluate the clinical performance of HCO3- assessment with this analyser in a real-life hemodialysis setting. Imprecision was calculated at different HCO3- levels, along with assay comparison with Gem Premier 4000 analysers. GEM Premier ChemSTAT displayed an imprecision and a bias (in comparison to GEM Premier 4000) for HCO3- of 0.4% and 37.3% at 20.8 mmol/L, 1.2% and 25.6% at 16.4 mmol/L, and 2.1% and 11.6% at 11.5 mmol/L, respectively, using three levels of HCO3- quality control sample ChemSTAT System Evaluator. At direct comparison with the GEM Premier 4000 in the hemodialysis setting, Bland-Altman analysis of HCO3- levels evidenced a bias (µ) of -4.9 (95% CI, -5.2 to -4.7) mmol/L. Such difference was attenuated by recalculating the GEM ChemSTAT expected HCO3- values from pH and pCO2 using the Henderson Hasselbach equation, µ=-0.07 (95%CI, -0.19 to 0.05) mmol/L (p = .24). In conclusion, our results show a remarkable difference between the HCO3- values reported by GEM ChemSTAT or GEM 4000. New reference values for GEM ChemSTAT HCO3- shall hence be defined according to our findings. We suggest that further investigation and a re-evaluation of the reference range should be made before extending the clinical use of this device.

The role of D-dimer in periprosthetic joint infection: a systematic review and meta-analysis.

OBJECTIVES: The current literature on diagnosis of periprosthetic joint infection (PJI) provides controversial evidence on the diagnostic accuracy of D-dimer. Therefore, this critical literature search and meta-analysis was aimed to summarize the diagnostic accuracy of D-dimer for diagnosing PJI. CONTENT: We searched MEDLINE, Scopus, and Web of Science, for studies on D-dimer for diagnosing PJI, according to the PRISMA flowchart. QUADAS was used for assessing study quality. Sensitivity, specificity, positive (PLR) and negative likelihood ratio (NLR), and diagnostic odds ratio (DOR) were analyzed using bivariate diagnostic random-effects model. The area under the receiver-operating curve (AUC-ROC) was calculated. Subgroup analysis and univariate meta-regression were carried out for detecting potential sources of heterogeneity. SUMMARY: We included 12 articles, totaling 1,818 patients (539 with PJI). The pooled sensitivity and specificity of D-dimer for diagnosing PJI were 0.739 (95% CI: 0.616-0.833) and 0.785 (95% CI: 0.679-0.863). The pooled PLR, NLR, DOR were 3.359 (95% CI, 2.340-4.821), 0.295 (95% CI, 0.180-0.484), and 11.787 (95% CI, 5.785-24.018). The cumulative ROC plot displayed an AUC of 0.688 (95% CI, 0.663-0.713; p<0.001). No threshold effects could be observed. The type of blood sample was identified as possible source of heterogeneity for DOR (p=0.01). OUTLOOK: Evidence emerged from this meta-analysis suggests that D-dimer displays sufficient diagnostic accuracy to rule out PJI. The type of blood sample (plasma vs. serum) and the study design could influence the results in terms of DOR and sensitivity. However, further perspective studies would be needed to validate its potential diagnostic usefulness.

Routine coagulation testing in Vacutainer® Citrate Plus tubes filled at minimum or optimal volume.

Background Filling of citrate tubes with appropriate amount of blood is essential for obtaining reliable results of coagulation testing. This study aimed to verify whether results of routine coagulation tests are comparable when the new Becton Dickinson Vacutainer® Citrate Plus tubes are filled at minimum or optimal volume. Methods The study population consisted of 133 patients (40 on oral anticoagulant therapy), who had blood collected for routine coagulation testing. Two sequential Vacutainer® Citrate Plus tubes of the same type and lot were drawn. The first tube was collected after a butterfly needle was inserted into the vein, so that the air in the tubing was aspirated into the tube before blood (minimum fill volume), whilst the second was drawn at optimal fill volume. Experiments were repeated using 2.7-mL (n = 86) and 1.8-mL (n = 47) tubes. Results Prothrombin time (PT) and fibrinogen values were slightly but significantly decreased in tubes with minimum than in those with optimal fill volume. The activated partial thromboplastin time (APTT) was slightly prolonged in tubes with minimum than in those with optimal fill volume, but the difference was not statistically significant. An identical trend was noted in separate analyses for the 2.7-mL and 1.8-mL tubes. Spearman's correlations between the two fill volumes were always >0.94 and bias was always within the quality specifications. Conclusions Blood drawing into Vacutainer® Citrate Plus tubes at minimum fill volume does not clinically bias routine coagulation testing.

[PHYSICAL, CHEMICAL AND MORPHOLOGICAL URINE EXAMINATION: RECOMMENDATIONS FOR THE POST ANALYTICAL PHASE FROM THE INTERDISCIPLINARY URINALYSIS GROUP (GIAU)].

With these recommendations the Interdisciplinary Urinalysis Group (GIAU) aims to stimulate the following aspects : improvement and standardization of the post analytical approach to physical, chemical and morphological urine examination (ECMU); emphasize the value added to ECMU by selection of clinically significant parameters, indication of analytical methods, of units of measurement, of reference values; improvement of interpretation of dip stick urinalysis with particular regard to the reconsideration of the diagnostic significance of the evaluated parameters together with an increasing awareness of the limits of sensitivity and specificity of this analytical method. Accompanied by the skills to propose and carry out in-depth investigations with analytical methods that are more sensitive and specific;increase the awareness of the importance of professional skills in the field of urinary morphology and their relationships with the clinicians. through the introduction, in the report, of descriptive and interpretative comments depending on the type of request, the complexity of the laboratory, the competence of the pathologist;implement a policy of evaluation of the analytical quality by using, in addition to traditional internal and external controls, a program for the evaluation of morphological competence. The hope is to revalue the enormous potential diagnostic of ECMU, implementing a urinalysis on personalized diagnostic needs that each patient brings with it.

Short preheating at 41°C leads to a red blood cells count comparable to that in RET channel of Sysmex analysers in samples showing cold agglutination.

AIMS: The presence of cold agglutinin in blood samples can cause a spontaneous agglutination of red blood cells (RBCs) when low temperature occurs. This phenomenon causes a spurious lowering of RBC count on the automated haematological analysers that are detected by incongruous values (≥370 g/L) of the mean cellular haemoglobi concentration (MCHC). A preheating at 37°C can remove the RBC agglutination generally resulting in a reliable count. It has been reported that the same result can be reached by using the optical reticulocyte (RET) channel of Sysmex analysers where the RBC count is not influenced by the presence of cold agglutinin. This study aims to evaluate these data in a larger population, with regard to environmental conditions on Sysmex analysers. We have also evaluated the influence of different thermal pretreatments on the RBC count. METHODS: This study was performed on 96 remnants of peripheral blood samples (48 with MCHC in normal range and 48 with MCHC>370 g/L) which have been analysed in different preanalytical conditions on the Sysmex analysers. RESULTS: A preheating of samples at 41°C for 1 min leads to a reversibility of the cold agglutination comparable to the one observed in the RET channel and yields better results compared with 37°C for 2 hours. CONCLUSIONS: None of described procedures assure the complete cold agglutination reversibility in every case. Consequently, since the haematological analysers not yet provide reliable parameters to confirm the complete resolution of agglutination, further verification of RBC count accuracy needs to be performed.

Automatic wedge smears preparation may cause traumatic morphological changes in peripheral blood cells.

In recent years, several automated analysers that prepare and stain blood smears have been introduced in clinical laboratories. Despite the use of instrumental settings based on physical characteristic of individual samples, traumatic injuries of neutrophil and lymphocytes can be observed. Some samples present a very high percentage of damaged cells, allowing the speculation that a cellular susceptibility may enhance mechanical traumatism. These artefacts can puzzle morphological evaluation in both traditional and digitised microscopy; in addition, unskilled operators can be misled.

Diagnostic accuracy of synovial fluid, blood markers, and microbiological testing in chronic knee prosthetic infections.

INTRODUCTION: This retrospective study was undertaken to define cut-off values for synovial fluid (SF) leukocyte count and neutrophil percentage for differentiating aseptic failure and periprosthetic joint infection (PJI) and to evaluate the diagnostic accuracy of blood inflammatory markers, and microbiological testing according to the criteria proposed by the International Consensus Meeting (ICM) of Philadelphia. METHODS: All patients who underwent revision total knee arthroplasty from January 2010 to July 2015 were included: we identified and classified 31 PJIs and 136 aseptic joints. The diagnostic performance of single test was assessed by receiver operating characteristic curve analyses. The sensitivity and specificity were calculated for each of the cut-off values and the area under the curve (AUC) was calculated. RESULTS: The median SF leukocyte count as well as the neutrophil percentage and inflammatory markers were significantly higher in patients with PJI than in those with aseptic failure (p < 0.001). A leukocyte count of > 2.8 × 103/µL had a sensitivity of 83.8% and a specificity of 89.7% whereas a neutrophil percentage of > 72% yielded a marginally higher sensitivity of 84% and a specificity of 91%. Applying the ICM criteria we found a significant correlation between all these diagnostic measures and PJI (p < 0.001) except for a single positive culture. The most accurate criterion of the ICM was the synovial neutrophil differential (AUC = 0.89; 95% CI 0.81-0.97), followed by SF leukocyte count (AUC = 0.86; 95% CI 0.78-0.94), increased inflammatory markers (AUC = 0.85; 95% CI 0.76-0.93), and two positive periprosthetic cultures (AUC = 0.84; 95% CI 0.73-0.94). The presence of sinus tract communicating with the joint and a single positive culture showed unfavourable diagnostic accuracy (AUC = 0.60, 95% CI 0.47-0.72; AUC = 0.49, 95% CI 0.38-0.61, respectively) CONCLUSIONS: The present study highlights the adequate ability of fluid cell count and neutrophil differential to distinguish between PJI and aseptic loosening. The clinical utility of fluid analysis in diagnosing infection can be improved by evaluation of other diagnostic criteria. LEVEL OF EVIDENCE: Level I Diagnostic Study.

Glucose variation in centrifuged serum and lithium-heparin gel tubes stored for up to 96 hours at room temperature or 4 °C.

This study aimed to verify glucose stability within centrifuged serum and lithium-heparin tubes stored at room temperature (RT) or 4 °C. Sixty paired serum (plus gel separator), lithium-heparin (plus gel separator) and K2-EDTA tubes were centrifuged within 30 min from collection. Thirty serum and lithium-heparin tubes were then stored at RT, whilst the other 30 serum and lithium-heparin tubes were kept at 4 °C. Complete cell blood count was performed in serum and plasma after centrifugation, as well as in K2-EDTA paired whole blood tubes. Glucose was measured immediately after centrifugation and 3, 6, 24, 48, 72 and 96 h afterwards. Immeasurable blood cells values were found in serum, whilst residual leukocytes and platelets were present in lithium-heparin plasma. Regardless of storage conditions, glucose concentration decreased 3 h after centrifugation in lithium-heparin tubes, displaying uninterrupted reduction until 96 h. Mean decrease per hour was higher in plasma tubes stored at RT than at 4 °C. Performance specification was exceeded between 6 and 24 h of storage in most plasma tubes. Glucose concentration significantly decreased in serum tubes between 24 and 48 h, regardless of storage conditions. The mean glucose variation never exceeded performance specification throughout the study period. Mean glucose decrease per hour in plasma was not associated with blood cells counts before and after centrifugation, and was probably attributable to the presence of blood cells entrapped within the gel. Delayed glucose measurement in centrifuged serum tubes may be clinically viable up to 96 h, whilst it may be unadvisable in centrifuged lithium-heparin tubes.

Analytical Evaluation of a Vancomycin Immunoassay in Synovial Fluid.

In clinical laboratories performing routine activities, the need to answer the burning clinical question in emerging field may be limited by lack of technology support or assays accessibility. Commercially available methods, although originally validated for specific biological matrices, may be employed for other matrices, following appropriate guidelines such as Clinical and Laboratory Standards Institute (CLSI) EP 19. We investigated the use of a vancomycin assay with synovial fluid samples, in view of a possible employment in vancomycin release study. The standard of care of periprosthetic joint infection is a two-stage revision surgery with antibiotic-loaded bone cement implantation. Vancomycin, for its activity against gram-positive bacteria even multidrug-resistant staphylococci, is the most widely used antibiotic. Despite the widespread use of such devices, little is known about the in vivo elution in the joint space. Clinical laboratories equipped with a validated, affordable method to quantify vancomycin in synovial fluid, may support clinical research, and give an important contribution to the study of the pharmacokinetics of antibiotic release from bone cement matrix.

Optimization of Cellular analysis of Synovial Fluids by optical microscopy and automated count using the Sysmex XN Body Fluid Mode.

BACKGROUND: This study was planned to assess the impact of pre-treating synovial fluid (SF) samples with hyaluronidase (HY), defining the best procedure for optical microscopy (OM) analysis and evaluating the performance of Sysmex XN-9000 Body Fluid module (XN-BF). METHODS: The cell count by OM was carried out both with and without HY pre-treatment, and using 3 different types of staining reagents. The evaluation of XN-BF included data comparison with OM (100 SFs), carryover, Limit of Blank (LoB), Limit of Detection (LoD), Limit of Quantitation (LoQ) and linearity. RESULTS: Unlike cell count in Burker's chamber and staining with Stromatol, pre-treatment with HY and staining with Methylene Blue and Turk's promoted cell clustering. The SF samples pre-treated with HY displayed excellent morphological quality, contrary to samples without HY pre-treatment. Excellent correlation was found between total cells counting with both OM and XN-BF. Satisfactory agreement was also observed between polymorphonuclear neutrophils compared to XN-BF parameter, whereas mononuclear cell count on XN-BF had suboptimal agreement with OM. The carryover was negligible. The LoB, LoD, LoQ and linearity were excellent. CONCLUSION: XN-BF displays excellent performance, which makes it a reliable and practical alternative to OM for SF samples analysis in clinical laboratories.