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20 LC-MS/MS 21 Vancomycin 22Tandem mass spectrometry 23Therapeutic drug monitoring 24 Background: Accurate quantification of vancomycin in plasma is important for adequate dose-adjustment.25 As literature suggests between-method differences, our first objective was to develop a novel liquid 26 chromatography-tandem mass spectrometry (LC-MS/MS) method for total vancomycin in human plasma and to 27 compare frequently used immunoassays with this method. Secondly, we investigated the clinical impact of 28 between-method quantification differences.29 Methods: For LC-MS/MS, lithium heparin plasma was extracted by adding a precipitation reagent containing the in-30 ternal standard (vancomycin-des-leucine). Analysis was performed on an Acquity TQD mass spectrometer 31 equipped with an Acquity UPLC 2795 separations module. Our method was analytically validated and compared 32 with four frequently used immunoassays from four different manufacturers. Vancomycin concentrations were 33 clinically classified as toxic, therapeutic and sub-therapeutic. Clinical discordance was calculated using LC-MS/MS 34 as a reference.35 Results: A novel LC-MS/MS method using protein precipitation as sole pretreatment and an analysis time of 5.0 min 36 was developed. The assay had a total imprecision of 2.6-8.5%, a limit of quantification of 0.3 mg/L and an accuracy 37 ranging from 101.4 to 111.2%. Using LC-MS/MS as reference, three immunoassays showed a mean proportional 38 difference within 10% and one showed a substantial mean proportional difference of N 20%. Clinical discordant 39 interpretation of the obtained concentrations ranged from 6.1 to 22.2%. 40 Conclusions: We developed a novel LC-MS/MS method for rapid analysis of total vancomycin concentrations in 41 human plasma. Correlation of the method with immunoassays showed a mean proportional difference N20% for 42 one of the assays, causing discordant clinical interpretation in more than 1 out of 5 samples.Q4 43

BackgroundImmunocompromised patients are at increased risk of severe COVID-19 and impaired vaccine response. In this observational prospective study, we evaluated immunogenicity of the BNT162b2 mRNA vaccine in cohorts of primary or secondary immunocompromised patients.MethodsFive clinical groups of immunocompromised patients [primary immunodeficiency (PID) (n=57), people living with HIV (PLWH) (n=27), secondary immunocompromised patients with a broad variety of underlying rheumatologic (n=23) and homogeneous (multiple sclerosis) neurologic (n=53) conditions and chronic kidney disease (CKD) (n=39)] as well as a healthy control group (n=54) were included. Systemic humoral and cellular immune responses were evaluated by determination of anti-SARS-CoV-2 Spike antibodies using a TrimericS IgG assay (Diasorin) and through quantification of interferon gamma release in response to SARS-CoV-2 antigen with QuantiFERON SARS-CoV-2 assay (Qiagen), respectively. Responses were measured at pre-defined time-points after complete vaccination.ResultsAll healthy controls, PLWH and CKD-patients had detectable antibodies 10 to 14 days (T2) and 3 months (T3) after administration of the second vaccination. In contrast, only 94.5% of the PID, 50.0% of the rheumatologic and 48.0% of neurologic patients developed antibodies at T2 and only 89.1% of the PID, 52.4% of the rheumatologic and 50.0% of neurologic patients developed antibodies at T3. At T3 no significant differences in cellular response between the healthy control group and the PLWH and CKD groups were found, while proportions of reactive subjects were lower in PID and rheumatologic patients and higher in neurologic patients. Humoral and cellular immune responses significantly correlated in the healthy control, PID, PLWH groups for all 3 antigens.ConclusionPatients with acquired or inherited immune disorders may show variable immune responses to vaccination with the BNT162b2 mRNA vaccine against SARS-CoV-2. Whether humoral, cellular or both immune responses are delayed depends on the patient group, therapy and individual risk factors. These data may guide the counselling of patients with immune disorders regarding vaccination of SARS-CoV-2.

New technological advances have paved the way for significant progress in automated urinalysis. Quantitative reading of urinary test strips using reflectometry has become possible, while complementary metal oxide semiconductor (CMOS) technology has enhanced analytical sensitivity and shown promise in microalbuminuria testing. Microscopy-based urine particle analysis has greatly progressed over the past decades, enabling high throughput in clinical laboratories. Urinary flow cytometry is an alternative for automated microscopy, and more thorough analysis of flow cytometric data has enabled rapid differentiation of urinary microorganisms. Integration of dilution parameters (e.g., creatinine, specific gravity, and conductivity) in urine test strip readers and urine particle flow cytometers enables correction for urinary dilution, which improves result interpretation. Automated urinalysis can be used for urinary tract screening and for diagnosing and monitoring a broad variety of nephrological and urological conditions; newer applications show promising results for early detection of urothelial cancer. Concomitantly, the introduction of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) has enabled fast identification of urinary pathogens. Automation and workflow simplification have led to mechanical integration of test strip readers and particle analysis in urinalysis. As the information obtained by urinalysis is complex, the introduction of expert systems may further reduce analytical errors and improve the quality of sediment and test strip analysis. With the introduction of laboratory-on-a-chip approaches and the use of microfluidics, new affordable applications for quantitative urinalysis and readout on cell phones may become available. In this review, we present the main recent developments in automated urinalysis and future perspectives.

We retrospectively compared the long-term evolution of IgG anti-spike (S) and anti-nucleocapsid (N) levels (Abbott immunoassays) in 116 non-severe and 115 severe SARS-CoV-2 infected patients from 2 university hospitals up to 365 days post positive RT-PCR. IgG anti-S and anti-N antibody levels decayed exponentially up to 365 days after a peak 0-59 days after positive RT-PCR. Peak antibody level/cut-off ratio 0-59 days after positive RT-PCR was more than 70 for anti-S compared to less than 6 for anti-N (p<0.01). Anti-S and anti-N were significantly higher in severe compared to non-severe patients up to 180-239 days and 300-365 days, respectively (p<0.05). Despite similar half-lives, the estimated time to 50% seronegativity was more than 2 years for anti-S compared to less than 1 year for anti-N in non-severe and severe COVID-19 patients, due to the significantly higher peak antibody level/cut-off ratio for anti-S compared to anti-N.