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.

External quality assessment providers' services appear to more impact the immunohaematology performance of laboratories than national regulatory and economic conditions.

OBJECTIVES: Medical laboratories may, at their own discretion, exceed but not undercut regulatory quality requirements. Available economic resources, however, may drive or hinder eagerness to exceed minimum requirements. Depending on the respective scopes of regulatory and economic framework conditions, differing levels of quality efforts to safeguard laboratory performance can be anticipated. However, this has not yet been investigated. METHODS: Immunohaematology external quality assessment (EQA) results collected by 26 EQA providers from their participant laboratories in 73 countries from 2004 to 2019 were evaluated. Error rates were aggregated in groups according to the respective national regulatory and economic framework conditions, to whether or not expert advice was provided in case of incorrect results, and the frequency of EQA samples. RESULTS: These representative data indicate no association between national regulatory (mandatory participation in EQA, monitoring of performance of individual laboratories by authorities, financial consequences of incorrect results) and economic (level of national income, share of national health expenditure) conditions to the quality performance of medical laboratories in immunohaematology. However, EQA providers' support for laboratories in the event of incorrect results appear to be associated with lower error rates, but a high EQA sample frequency with higher error rates. CONCLUSIONS: Further research into the impact of introducing or changing services of EQA providers is needed to confirm the results found in this first of its kind study.

Reperfusion Injury after ischemic Stroke Study (RISKS): single-centre (Florence, Italy), prospective observational protocol study.

INTRODUCTION: Treatments aiming at reperfusion of the acutely ischaemic brain tissue may result futile or even detrimental because of the so-called reperfusion injury. The processes contributing to reperfusion injury involve a number of factors, ranging from blood-brain barrier (BBB) disruption to circulating biomarkers. Our aim is to evaluate the relative effect of imaging and circulating biomarkers in relation to reperfusion injury. METHODS AND ANALYSIS: Observational hospital-based study that will include 140 patients who had ischaemic stroke, treated with systemic thrombolysis, endovascular treatment or both. BBB disruption will be assessed with CT perfusion (CTP) before treatment, and levels of a large panel of biomarkers will be measured before intervention and after 24 hours. Relevant outcomes will include: (1) reperfusion injury, defined as radiologically relevant haemorrhagic transformation at 24 hours and (2) clinical status 3 months after the index stroke. We will investigate the separate and combined effect of pretreatment BBB disruption and circulating biomarkers on reperfusion injury and clinical status at 3 months. Study protocol is registered at http://www.clinicaltrials.gov (ClinicalTrials.gov ID: NCT03041753). ETHICS AND DISSEMINATION: The study protocol has been approved by ethics committee of the Azienda Ospedaliero Universitaria Careggi (Università degli Studi di Firenze). Informed consent is obtained by each patient at time of enrolment or deferred when the participant lacks the capacity to provide consent during the acute phase. Researchers interested in testing hypotheses with the data are encouraged to contact the corresponding author. Results from the study will be disseminated at national and international conferences and in medical thesis. TRIAL REGISTRATION NUMBER: NCT03041753.

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.

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.

Biochemical markers in the follow-up of the osteoporotic patients.

Osteoporosis, a disease characterised by low bone mass and micro-architectural deterioration of bone tissue, is viewed as an emerging medical condition. Bone mineral density (BMD) is considered the gold standard of bone status assessment, however it does not offer the timely response desirable for monitoring. Biochemical markers of bone turnover (BTMs) are claimed to be suitable for that purpose. There is not generalized agreement on which marker could be used in routine. The present paper reviews pros and cons of currently used BTMs and relative analytical methods. Several analytical issues, such as biological variability, molecules stability, lack of reference materials jeopardize the field and, consequently, recommendations are difficult to be drawn. Reference range can't be used to support clinical judgement and, in this view, Least Significant Change (LSC) is regarded as a way to improve the interpretation of analytical results.Bone alkaline phosphatase (bALP) is still a marker of interest and its use is widespread in clinical laboratories; Tartrate Resistant Acid Phosphatase band 5b (TRAP 5b) appears to be a promising marker. N-terminal propeptides of type I collagen (s-PINP) and beta-collagen 1 C-terminal cross linked telopeptides (s-CTX), given low biological variability and assay availability for automatised instruments, should be the marker of choice in future clinical trials, to overcome the paucity of uniform data and should be used in clinical routine, to monitor osteoporosis treatment. Finally, the lack of standardisation of currently available diagnostic methods, could be overcome by harmonisation.

Lack of concordance between the 75-g and 100-g glucose load tests for the diagnosis of gestational diabetes mellitus.

BACKGROUND: Gestational diabetes mellitus (GDM) is common and can have a substantial impact on fetal growth, birth weight, and morbidity. The American Diabetes Association recommends GDM testing with either a 3-h, 100-g glucose load (100 g) (criteria according to Am J Obstet Gynecol 1982;144:768-73) or a 2-h, 75-g glucose load (75g). We investigated the comparability of the 75 g and the 100g tests in the diagnosis of GDM. METHODS: From January 1997 to December 1999, in 1061 consecutive Caucasian nonobese and nondiabetic pregnant women who attended the Maternal-Fetal Medicine Unit, we performed GDM testing with a 75-g load during 2 periods of pregnancy: early (16-20 weeks) and late (26-30 weeks). Because we assumed there would be few GBM cases in women with a 1-h plasma glucose <1300 mg/L in the 75 g test, we did not retest these women. We retested the remaining women with possible or diagnosed GDM with a 100-g load within a week. RESULTS: GDM was diagnosed in 41 of 227 women with the 100-g load and 15 of 227 with the 75-g load (11 concordant); the kappa index was 0.21. At 26-31 weeks of pregnancy, 484 of 976 women (49.9%) underwent both tests. GDM was diagnosed in 60 of 484 woman with the 100-g load and in 26 of 484 with the 75-g load (13 concordant); the kappa index was 0.18. CONCLUSIONS: Among women with possible GDM in both early and late periods of pregnancy, there was only weak diagnostic agreement between results determined with 75-g and 100-g glucose loads.